Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays

ABSTRACT

The present invention describes a method for identifying one or more of a plurality of sequences differing by one or more single base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences. The method includes a ligation phase, a capture phase, and a detection phase. The ligation phase utilizes a ligation detection reaction between one oligonucleotide probe, which has a target sequence-specific portion and an addressable array-specific portion, and a second oligonucleotide probe, having a target sequence-specific portion and a detectable label. After the ligation phase, the capture phase is carried out by hybridizing the ligated oligonucleotide probes to a solid support with an array of immobilized capture oligonucleotides at least some of which are complementary to the addressable array-specific portion. Following completion of the capture phase, a detection phase is carried out to detect the labels of ligated oligonucleotide probes hybridized to the solid support.

The present application is a continuation of U.S. patent applicationSer. No. 08/794,851, filed Feb. 4, 1997, now U.S. Pat. No. 6,852,487,issued on Feb. 8, 2005, which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 60/011,359, filed Feb. 9, 1996.

This invention was made with government support under grant numberGM-41337-06 awarded by National Institutes of Health. The U.S.Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the detection of nucleic acid sequencedifferences in nucleic acids using a ligation phase, a capture phase,and a detection phase. The ligation phase utilizes a ligation detectionreaction between one oligonucleotide probe which has a targetsequence-specific portion and an addressable array-specific portion anda second oligonucleotide probe having a target sequence-specific portionand a detectable-label. The capture phase-involves hybridizing theligated oligonucleotide probes to a solid support with an array ofimmobilized capture oligonucleotides at least some of which arecomplementary to the addressable array-specific portion. The labels ofligated oligonucleotide probes hybridized to the solid support aredetected during the detection phase.

BACKGROUND OF THE INVENTION Detection of Sequence Differences

Large-scale multiplex analysis of highly polymorphic loci is needed forpractical identification of individuals, e.g., for paternity testing andin forensic science (Reynolds et al., Anal. Chem., 63:2-15 (1991)), fororgan-transplant donor-recipient matching (Buyse et al., TissueAntigens, 41:1-14 (1993) and Gyllensten et al., PCR Meth. Appl, 1:91-98(1991)), for genetic disease diagnosis, prognosis, and pre-natalcounseling (Chamberlain et al., Nucleic Acids Res., 16:11141-11156(1988) and L. C. Tsui, Human Mutat., 1:197-203 (1992)), and the study ofoncogenic mutations (Hollstein et al., Science, 253:49-53 (1991)). Inaddition, the cost-effectiveness of infectious disease diagnosis bynucleic acid analysis varies directly with the multiplex scale in paneltesting. Many of these applications depend on the discrimination ofsingle-base differences at a multiplicity of sometimes closely spaceloci.

A variety of DNA hybridization techniques are available for detectingthe presence of one or more selected polynucleotide sequences in asample containing a large number of sequence regions. In a simplemethod, which relies on fragment capture and labeling, a fragmentcontaining a selected sequence is captured by hybridization to animmobilized probe. The captured fragment can be labeled by hybridizationto a second probe which contains a detectable reporter moiety.

Another widely used method is Southern blotting. In this method, amixture of DNA fragments in a sample are fractionated by gelelectrophoresis, then fixed on a nitrocellulose filter. By reacting thefilter with one or more labeled probes under hybridization conditions,the presence of bands containing the probe sequence can be identified.The method is especially useful for identifying fragments in arestriction-enzyme DNA digest which contain a given probe sequence, andfor analyzing restriction-fragment length polymorphisms (“RFLPs”).

Another approach to detecting the presence of a given sequence orsequences in a polynucleotide sample involves selective amplification ofthe sequence(s) by polymerase chain reaction. U.S. Pat. No. 4,683,202 toMullis, et al. and R. K. Saiki, et al., Science 230:1350 (1985). In thismethod, primers complementary to opposite end portions of the selectedsequence(s) are used to promote, in conjunction with thermal cycling,successive rounds of primer-initiated replication. The amplifiedsequence may be readily identified by a variety of techniques. Thisapproach is particularly useful for detecting the presence of low-copysequences in a polynucleotide-containing sample, e.g., for detectingpathogen sequences in a body-fluid sample.

More recently, methods of identifying known target sequences by probeligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M.Whiteley, et al., D. Y. Wu, et al., Genomics 4:560 (1989), U. Landegren,et al., Science 241:1077 (1988), and E. Winn-Deen, et al., Clin. Chem.37:1522 (1991). In one approach, known as oligonucleotide ligation assay(“OLA”), two probes or probe elements which span a target region ofinterest are hybridized with the target region. Where the probe elementsmatch (basepair with) adjacent target bases at the confronting ends ofthe probe elements, the two elements can be joined by ligation, e.g., bytreatment with ligase. The ligated probe element is then assayed,evidencing the presence of the target sequence.

In a modification of this approach, the ligated probe elements act as atemplate for a pair of complementary probe elements. With continuedcycles of denaturation, hybridization, and ligation in the presence ofthe two complementary pairs of probe elements, the target sequence isamplified geometrically, i.e., exponentially allowing very small amountsof target sequence to be detected and/or amplified. This approach isreferred to as ligase chain reaction (“LCR”). F. Barany, “GeneticDisease Detection and DNA Amplification Using Cloned ThermostableLigase,” Proc. Nat'l Acad. Sci. USA, 88:189-93 (1991) and F. Barany,“The Ligase Chain Reaction (LCR) in a PCR World,” PCR Methods andApplications, 1:5-16 (1991).

Another scheme for multiplex detection of nucleic acid sequencedifferences is disclosed in U.S. Pat. No. 5,470,705 to Grossman et. al.where sequence-specific probes, having a detectable label and adistinctive ratio of charge/translational frictional drag, can behybridized to a target and ligated together. This technique was used inGrossman, et. al., “High-density Multiplex Detection of Nucleic AcidSequences: Oligonucleotide Ligation Assay and Sequence-codedSeparation,” Nucl. Acids Res. 22(21):4527-34 (1994) for the large scalemultiplex analysis of the cystic fibrosis transmembrane regulator gene.

Jou, et. al., “Deletion Detection in Dystrophin Gene by Multiplex GapLigase Chain Reaction and Immunochromatographic Strip Technology,” HumanMutation 5:86-93 (1995) relates to the use of a so called “gap ligasechain reaction” process to amplify simultaneously selected regions ofmultiple exons with the amplified products being read on animmunochromatographic strip having antibodies specific to the differenthaptens on the probes for each exon.

There is a growing need, e.g., in the field of genetic screening, formethods useful in detecting the presence or absence of each of a largenumber of sequences in a target polynucleotide. For example, as many as400 different mutations have been associated with cystic fibrosis. Inscreening for genetic predisposition to this disease, it is optimal totest all of the possible different gene sequence mutations in thesubject's genomic DNA, in order to make a positive identification of“cystic fibrosis”. It would be ideal to test for the presence or absenceof all of the possible mutation sites in a single assay. However, theprior-art methods described above are not readily adaptable for use indetecting multiple selected sequences in a convenient, automatedsingle-assay format.

Solid-phase hybridization assays require multiple liquid-handling steps,and some incubation and wash temperatures must be carefully controlledto keep the stringency needed for single-nucleotide mismatchdiscrimination. Multiplexing of this approach has proven difficult asoptimal hybridization conditions vary greatly among probe sequences.

Allele-specific PCR products generally have the same size, and a givenamplification tube is scored by the presence or absence of the productband in the gel lane associated with each reaction tube. Gibbs et al.,Nucleic Acids Res., 17:2437-2448 (1989). This approach requiressplitting the test sample among multiple reaction tubes with differentprimer combinations, multiplying assay cost. PCR has also discriminatedalleles by attaching different fluorescent dyes to competing allelicprimers in a single reaction tube (F. F. Chehab, et al., Proc. Natl.Acad. Sci. USA, 86:9178-9182 (1989)), but this route to multiplexanalysis is limited in scale by the relatively few dyes which can bespectrally resolved in an economical manner with existinginstrumentation and dye chemistry. The incorporation of bases modifiedwith bulky side chains can be used to differentiate allelic PCR productsby their electrophoretic mobility, but this method is limited by thesuccessful incorporation of these modified bases by polymerase, and bythe ability of electrophoresis to resolve relatively large PCR productswhich differ in size by only one of these groups. Livak et al., NucleicAcids Res., 20:4831-4837 (1989). Each PCR product is used to look foronly a single mutation, making multiplexing difficult.

Ligation of allele-specific probes generally has used solid-phasecapture (U. Landegren et al., Science, 241:1077-1080 (1988); Nickersonet al., Proc. Natl. Acad. Sci. USA, 87:8923-8927 (1990)) orsize-dependent separation (D. Y. Wu, et al., Genomics, 4:560-569 (1989)and F. Barany, Proc. Natl. Acad. Sci., 88:189-193 (1991)) to resolve theallelic signals, the latter method being limited in multiplex scale bythe narrow size range of ligation probes. The gap ligase chain reactionprocess requires an additional step—polymerase extension. The use ofprobes with distinctive ratios of charge/translational frictional dragtechnique to a more complex multiplex will either require longerelectrophoresis times or the use of an alternate form of detection.

The need thus remains for a rapid single assay format to detect thepresence or absence of multiple selected sequences in a polynucleotidesample.

Use of Oligonucleotide Arrays for Nucleic Acid Analysis

Ordered arrays of oligonucleotides immobilized on a solid support havebeen proposed for sequencing, sorting, isolating, and manipulating DNA.It has been recognized that hybridization of a cloned single-strandedDNA molecule to all possible oligonucleotide probes of a given lengthcan theoretically identify the corresponding complementary DNA segmentspresent in the molecule. In such an array, each oligonucleotide probe isimmobilized on a solid support at a different predetermined position.All the oligonucleotide segments in a DNA-molecule can be surveyed withsuch an array.

One example of a procedure for sequencing DNA molecules using arrays ofoligonucleotides is disclosed in U.S. Pat. No. 5,202,231 to Drmanac, et.al. This involves application of target DNA to a solid support to whicha plurality of oligonucleotides are attached. Sequences are read byhybridization of segments of the target DNA to the oligonucleotides andassembly of overlapping segments of hybridized oligonucleotides. Thearray utilizes all possible oligonucleotides of a certain length between11 and 20 nucleotides, but there is little information about how thisarray is constructed. See also A. B. Chetverin, et. al., “Sequencing ofPools of Nucleic Acids on Oligonucleotide Arrays,” BioSystems 30: 215-31(1993); WO 92/16655 to Khrapko et. al.; Kuznetsova, et. al., “DNASequencing by Hybridization with Oligonucleotides Immobilized in Gel.Chemical Ligation as a Method of Expanding the Prospects for theMethod,” Mol. Biol. 28(20): 290-99(1994); M. A. Livits, et. al.,“Dissociation of Duplexes Formed by Hybridization of DNA withGel-Immobilized Oligonucleotides,” J. Biomolec. Struct. & Dynam. 11(4):783-812 (1994).

WO 89/10977 to Southern discloses the use of a support carrying an arrayof oligonucleotides capable of undergoing a hybridization reaction foruse in analyzing a nucleic acid sample for known point mutations,genomic fingerprinting, linkage analysis, and sequence determination.The matrix is formed by laying nucleotide bases in a selected pattern onthe support. This reference indicates that a hydroxyl linker group canbe applied to the support with the oligonucleotides being assembled by apen plotter or by masking.

WO 94/11530 to Cantor also relates to the use of an oligonucleotidearray to carry out a process of sequencing by hybridization. Theoligonucleotides are duplexes having overhanging ends to which targetnucleic acids bind and are then ligated to the non-overhanging portionof the duplex. The array is constructed by using streptavidin-coatedfilter paper which captures biotinylated oligonucleotides assembledbefore attachment.

WO 93/17126 to Chetverin uses sectioned, binary oligonucleotide arraysto sort and survey nucleic acids. These arrays have a constantnucleotide sequence attached to an adjacent variable nucleotidesequence, both bound to a solid support by a covalent linking moiety.The constant-nucleotide sequence has a priming region to permitamplification by PCR of hybridized strands. Sorting is then carried outby hybridization to the variable region. Sequencing, isolating, sorting,and manipulating fragmented nucleic acids on these binary arrays arealso disclosed. In one embodiment with enhanced sensitivity, theimmobilized oligonucleotide has a shorter complementary regionhybridized to it, leaving part of the oligonucleotide uncovered. Thearray is then subjected to hybridization conditions so that acomplementary nucleic acid anneals to the immobilized oligonucleotide.DNA ligase is then used to join the shorter complementary region and thecomplementary nucleic acid on the array. There is little disclosure ofhow to prepare the arrays of oligonucleotides.

WO 92/10588 to Fodor et. al., discloses a process for sequencing,fingerprinting, and mapping nucleic acids by hybridization to an arrayof oligonucleotides. The array of oligonucleotides is prepared by a verylarge scale immobilized polymer synthesis which permits the synthesis oflarge, different oligonucleotides. In this procedure, the substratesurface is functionalized and provided with a linker group by whicholigonucleotides are assembled on the substrate. The regions whereoligonucleotides are attached have protective groups (on the substrateor individual nucleotide subunits) which are selectively activated.Generally, this involves imaging the array with light using a mask ofvarying configuration so that areas exposed are deprotected. Areas whichhave been deprotected undergo a chemical reaction with a protectednucleotide to extend the oligonucleotide sequence where imaged. A binarymasking strategy can be used to build two or more arrays at a giventime. Detection involves positional localization of the region wherehybridization has taken place. See also U.S. Pat. Nos. 5,324,633 and5,424,186 to Fodor et. al., U.S. Pat. Nos. 5,143,854 and 5,405,783 toPirrung, et. al., WO 90/15070 to Pirrung, et. al., A. C. Pease, et. al.,“Light-generated Oligonucleotide Arrays for Rapid DNA SequenceAnalysis”, Proc. Natl. Acad. Sci USA 91: 5022-26 (1994). K. L. Beattie,et. al., “Advances in Genosensor Research,” Clin. Chem. 41(5): 700-09(1995) discloses attachment of previously assembled oligonucleotideprobes to a solid support.

There are many drawbacks to the procedures for sequencing byhybridization to such arrays. Firstly, a very large number ofoligonucleotides must be synthesized. Secondly, there is poordiscrimination between correctly hybridized, properly matched duplexesand those which are mismatched. Finally, certain oligonucleotides willbe difficult to hybridize to under standard conditions, with sucholigonucleotides being capable of identification only through extensivehybridization studies.

The present invention is directed toward overcoming these deficienciesin the art.

SUMMARY OF THE INVENTION

The present invention relates to a method for identifying one or more ofa plurality of sequences differing by one or more single base changes,insertions, deletions, or translocations in a plurality of targetnucleotide sequences. The method includes a ligation phase, a capturephase, and a detection phase.

The ligation phase requires providing a sample potentially containingone or more nucleotide sequences with a plurality of sequencedifferences. A plurality of oligonucleotide sets are utilized in thisphase. Each set includes a first oligonucleotide probe, having atarget-specific portion and an addressable array-specific portion, and asecond oligonucleotide probe, having a target-specific portion and adetectable reporter label. The first and second oligonucleotide probesin a particular set are suitable for ligation together when hybridizedadjacent to one another on a corresponding target nucleotide sequence.However, the first and second oligonucleotide probes have a mismatchwhich interferes with such ligation when hybridized to anothernucleotide sequence present in the sample. A ligase is also utilized.The sample, the plurality of oligonucleotide probe sets, and the ligaseare blended to form a mixture. The mixture is subjected to one or moreligase detection reaction cycles comprising a denaturation treatment anda hybridization treatment. The denaturation treatment involvesseparating any hybridized oligonucleotides from the target nucleotidesequences. The hybridization treatment involves hybridizing theoligonucleotide probe sets at adjacent positions in a base-specificmanner to their respective target nucleotide sequences, if present inthe sample, and ligating them to one another to form a ligated productsequence containing (a) the addressable array-specific portion, (b) thetarget-specific portions connected-together, and (c) the detectablereporter label. The oligonucleotide probe sets may hybridize tonucleotide sequences in the sample other than their respective targetnucleotide sequences but do not ligate together due to a presence of oneor more mismatches and individually separate during denaturationtreatment.

The next phase of the process is the capture phase. This phase involvesproviding a solid support with capture oligonucleotides immobilized atparticular sites. The capture oligonucleotides are complementary to theaddressable array-specific portions. The mixture, after being subjectedto the ligation phase, is contacted with the solid support underconditions effective to hybridize the addressable array-specificportions to the capture oligonucleotides in a base-specific manner. As aresult, the addressable array-specific portions are captured on thesolid support at the site with the complementary captureoligonucleotides.

After the capture phase is the detection phase. During this portion ofthe process, the reporter labels of the ligated product sequences arecaptured on the solid support at particular sites. When the presence ofthe reporter label bound to the solid support is detected, therespective presence of one or more nucleotide sequences in the sample isindicated.

The present invention also relates to a kit for carrying out the methodof the present invention which includes the ligase, the plurality ofoligonucleotide sets, and the solid support with immobilized captureoligonucleotides.

Another aspect of the present invention relates to a method of formingan array of oligonucleotides on a solid support. This method involvesproviding a solid support having an array of positions each suitable forattachment of an oligonucleotide. A linker or surface (which can benon-hydrolyzable), suitable for coupling an oligonucleotide to the solidsupport at each of the array positions, is attached to the solidsupport. An array of oligonucleotides on a solid support is formed by aseries of cycles of activating selected array positions for attachmentof multimer nucleotides and attaching multimer nucleotides at theactivated array positions.

Yet another aspect of the present invention relates to an array ofoligonucleotides on a solid support per se. The solid support has anarray of positions each suitable for attachment of an oligonucleotide. Alinker or support (which can be non-hydrolyzable), suitable for couplingan oligonucleotide to the solid support, is attached to the solidsupport at each of the array positions. An array of oligonucleotides areplaced on a solid support with at least some of the array positionsbeing occupied by oligonucleotides having greater than sixteennucleotides.

The present invention contains a number of advantages over prior artsystems, particularly, its ability to carry out multiplex analyses ofcomplex genetic systems. As a result, a large number of nucleotidesequence differences in a sample can be detected at one time. Thepresent invention is useful for detection of, for example, cancermutations, inherited (germline) mutations, and infectious diseases. Thistechnology can also be utilized in conjunction with environmentalmonitoring, forensics, and the food industry.

In addition, the present invention provides quantitative detection ofmutations in a high background of normal sequences, allows detection ofclosely-clustered mutations, permits detection using addressable arrays,and is amenable to automation. By combining the sensitivity of PCR withthe specificity of LDR, common difficulties encountered inallele-specific PCR, such as false-positive signal generation, primerinterference during multiplexing, limitations in obtaining quantitativedata, and suitability for automation, have been obviated. In addition,by relying on the specificity of LDR to distinguish single-basemutations, the major inherent problem of oligonucleotide probe arrays(i.e. their inability to distinguish single-base changes at allpositions in heterozygous samples) has been overcome. PCR/LDR addressesthe current needs in cancer detection; to quantify mutations which mayserve as clonal markers and to detect minimal residual disease andmicrometastases.

In carrying out analyses of different samples, the solid supportcontaining the array can be reused. This reduces the quantity of solidsupports which need to be manufactured and lowers the cost of analyzingeach sample.

The present invention also affords great flexibility in the synthesis ofoligonucleotides and their attachment to solid supports.Oligonucleotides can be synthesized off of the solid support and thenattached to unique surfaces on the support. This technique can be usedto attach-full length oligonucleotides or peptide nucleotide analogues(“PNA”) to the solid support. Alternatively, shorter nucleotide oranalogue segments (dimer, trimer, tetramer, etc.) can be employed in asegment condensation or block synthesis approach to full lengtholigomers on the solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram depicting polymerase chain reaction(“PCR”)/ligase detection reaction (“LDR”) processes, according to theprior art and the present invention, for detection of germlinemutations, such as point mutations.

FIG. 2 is a flow diagram depicting PCR/LDR processes, according to theprior art and the present invention, for detection of cancer-associatedmutations.

FIG. 3 is a schematic diagram depicting a PCR/LDR process, according tothe present invention, using addresses on the allele-specific probes fordetecting homo- or heterozygosity at two polymorphisms (i.e. alleledifferences) on the same gene.

FIG. 4 is a schematic diagram depicting a PCR/LDR process, according tothe present invention, using addresses on the allele-specific probeswhich distinguishes all possible bases at a given site.

FIG. 5 is a schematic diagram depicting a PCR/LDR process, according tothe present invention, using addresses on the allele-specific probes fordetecting the presence of any possible base at two nearby sites.

FIG. 6 is a schematic diagram of a PCR/LDR process, according to thepresent invention, using addresses on the allele-specific probesdistinguishing insertions and deletions.

FIG. 7 is a schematic diagram of a PCR/LDR process, in accordance withthe present invention, using addresses on the allele-specific probes todetect a low abundance mutation (within a codon) in the presence of anexcess of normal sequence.

FIG. 8 is a schematic diagram of a PCR/LDR process, in accordance withthe present invention, where the address is placed on the common probeand the allele differences are distinguished by different fluorescentsignals F1, F2, F3, and F4.

FIG. 9 is a schematic diagram of a PCR/LDR process, in accordance withthe present invention, where both adjacent and nearby alleles aredetected.

FIG. 10 is a schematic diagram of a PCR/LDR process, in accordance withthe present invention, where all possible single-base mutations for asingle codon are detected.

FIG. 11 shows the chemical reactions for covalent modifications,grafting, and oligomer attachments to solid supports.

FIGS. 12A-C show proposed chemistries for covalent attachment ofoligonucleotides to solid supports.

FIGS. 13A-C show two alternative formats for oligonucleotide probecapture. In FIG. 13A, the addressable array-specific portions are shownon the allele-specific probe or the common probe. When the addressablearray-specific portions are on the allele-specific probe, alleles aredistinguished by capture of fluorescent signals on addresses Z1 and Z2,respectively, as shown in FIG. 13B. In FIG. 13C, the addressablearray-specific portions are on the common probe and alleles aredistinguished by capture of fluorescent signals F1 and F2, whichcorrespond to the two alleles, respectively.

FIGS. 14A-E depict a protocol for constructing an 8×8 array of oligomersby spotting full-length, individual 24 mer oligomers at various sites ona solid support.

FIGS. 15A-E are perspective views of the 8×8 array construction protocolof FIGS. 14A-E.

FIGS. 16A-C are views of an apparatus used to spot full-length,individual 24 mer oligomers on a solid support in accordance with FIGS.14A-E to 15A-E.

FIG. 17 shows a design in accordance with the present invention using 36tetramers differing by at least 2 bases, which can be used to create aseries of unique 24-mers.

FIG. 18A-G are schematic diagrams showing addition of PNA tetramers togenerate a 5×5 array of unique 25 mer addresses.

FIGS. 19A-E depict a protocol for constructing an 8×8 array of 24-mersby sequentially coupling 6 tetramers.

FIGS. 20A-C are perspective views of the 8×8 array construction protocolof FIGS. 19B-C.

FIGS. 21A-F show a schematic cross-sectional view of the synthesis of anaddressable array, in accordance with FIGS. 19B-C.

FIGS. 22A-C are schematic views of an apparatus used to synthesize the8×8 array of 24 mers on a solid support in accordance with FIGS. 19B-C,20A-C, and 21A-G.

FIGS. 23A-C are perspective views of the 8×8 array construction protocolof FIG. 19 (FIGS. 19D-19E).

FIGS. 24A-C are schematic views of an apparatus used to synthesize the5×5 array of 24 mers on a solid support, in accordance with FIGS. 19D-Eand 23A-C.

FIGS. 25A-C are schematic diagrams of a valve block assembly capable ofrouting six input solutions to 5 output ports.

FIGS. 26A-D are diagrams of a circular manifold capable ofsimultaneously channeling 6 input solutions into 5 output ports.

FIG. 27 is a schematic drawing of an assay system for carrying out theprocess of the present invention.

FIG. 28 shows phosphorimager data for different derivatized surfaces.

FIG. 29 shows phosphorimager data for different crosslinking conditionsof the polymer matrix.

FIG. 30 shows phosphorimager data for —OH functionalized slides.

FIG. 31 shows the reaction scheme for producing a glass slide silanizedwith 3-methacryloyloxypropyltrimethoxysilane.

FIG. 32 shows the reaction scheme for producing polymerizedpoly(ethylene glycol)methacrylate on a glass slide silanized with3-methacryloyloxypropyl-trimethoxy-silane.

FIG. 33 shows the reaction scheme for producing polymerized acrylic acidand trimethylolpropane ethoxylate-(14/3 EO/OH) triacrylate on a glassslide silanized with 3-methacryloyloxypropyltrimethoxysilane.

FIG. 34 shows the reaction scheme for producing polymerizedpoly(ethylene glycol)methacrylate and trimethylolpropane ethoxylate(14/3 EO/OH) triacrylate on a glass slide silanized with3-methacryloyloxypropyltrimethoxysilane.

DETAILED DESCRIPTION OF THE INVENTION AND DRAWINGS

The present invention relates to a method for identifying one or more ofa plurality of sequences differing by one or more single-base changes,insertions, deletions, or translocations in a plurality of targetnucleotide sequences. The method includes a ligation phase, a capturephase, and a detection phase.

The ligation phase requires providing a sample potentially containingone or more nucleotide sequences with a plurality of sequencedifferences. A plurality of oligonucleotide sets are utilized in thisphase. Each set includes a first oligonucleotide probe, having atarget-specific portion and an addressable array-specific portion, and asecond oligonucleotide probe, having a target-specific portion and adetectable reporter label. The first and second oligonucleotide probesin a particular set are suitable for ligation together when hybridizedadjacent to one another on a corresponding target nucleotide sequence.However, the first and second oligonucleotide probes have a mismatchwhich interferes with such ligation when hybridized to anothernucleotide sequence present in the sample. A ligase is also utilized.The sample, the plurality of oligonucleotide probe sets, and the ligaseare blended to form a mixture. The mixture is subjected to one or moreligase detection reaction cycles comprising a denaturation treatment anda hybridization treatment. The denaturation treatment involvesseparating any hybridized oligonucleotides from the target nucleotidesequences. The hybridization treatment involves hybridizing theoligonucleotide probe sets at adjacent positions in a base-specificmanner to their respective target nucleotide sequences, if present inthe sample, and ligating them to one another to form a ligated productsequence containing (a) the addressable array-specific portion, (b) thetarget-specific portions connected together, and (c) the detectablereporter label. The oligonucleotide probe sets may hybridize tonucleotide sequences in the sample other than their respective targetnucleotide sequences but do not ligate together due to a presence of oneor more mismatches and individually separate during denaturationtreatment.

The next phase of the process is the capture phase. This phase involvesproviding a solid support with capture oligonucleotides immobilized atparticular sites. The capture oligonucleotides are complementary to theaddressable array-specific portions. The mixture, after being subjectedto the ligation phase, is contacted with the solid support underconditions effective to hybridize the addressable array-specificportions to the capture oligonucleotides in a base-specific manner. As aresult, the addressable array-specific portions are captured on thesolid support at the site with the complementary captureoligonucleotides.

After the capture phase is the detection phase. During this portion ofthe process, the reporter labels of the ligated product sequences arecaptured on the solid support at particular sites. When the presence ofthe reporter label bound to the solid support is detected, therespective presence of one or more nucleotide sequences in the sample isindicated.

Often, a number of different single-base mutations, insertions, ordeletions may occur at the same nucleotide position of the sequence ofinterest. The method provides for having an oligonucleotide set, wherethe second oligonucleotide probe is common and contains the detectablelabel, and the first oligonucleotide probe has different addressablearray-specific portions and target-specific portions. The firstoligonucleotide probe is suitable for ligation to a second adjacentoligonucleotide probe at a first ligation junction, when hybridizedwithout mismatch, to the sequence in question. Different first adjacentoligonucleotide probes would contain different discriminating base(s) atthe junction where only a hybridization without mismatch at the junctionwould allow for ligation. Each first adjacent oligonucleotide wouldcontain a different addressable array-specific portion, and, thus,specific base changes would be distinguished by capture at differentaddresses. In this scheme, a plurality of different captureoligonucleotides are attached at different locations on the solidsupport for multiplex detection of additional nucleic acid sequencesdiffering from other nucleic acids by at least a single base.Alternatively, the first oligonucleotide probe contains commonaddressable array-specific portions, and the second oligonucleotideprobes have different detectable labels and target-specific portions.

Such arrangements permit multiplex detection of additional nucleic acidsequences differing from other nucleic acids by at least a single base.The nucleic acids sequences can be on the same or different alleles whencarrying out such multiplex detection.

The present invention also relates to a kit for carrying out the methodof the present invention which includes the ligase, the plurality ofdifferent oligonucleotide probe sets, and the solid support withimmobilized capture oligonucleotides. Primers for preliminaryamplification of the target nucleotide sequences may also be included inthe kit. If amplification is by polymerase chain reaction, polymerasemay also be included in the kit.

FIGS. 1 and 2 show flow diagrams of the process of the present inventioncompared to a prior art ligase detection reaction utilizing capillary orgel electrophoresis/fluorescent quantification. FIG. 1 relates todetection of a germline mutation detection, while FIG. 2 shows thedetection of cancer.

FIG. 1 depicts the detection of a germline point mutation, such as thep53 mutations responsible for Li-Fraumeni syndrome. In step 1, after DNAsample preparation, exons 5-8 are PCR amplified using Taq (i.e. Thermusaquaticus) polymerase under hot start conditions. At the end of thereaction, Taq polymerase is degraded by heating at 100° C. for 10 min.Products are diluted 20-fold in step 2 into fresh LDR buffer containingallele-specific and common LDR probes. A tube generally contains about100 to 200 moles of each primer. In step 3, the ligase detectionreaction is initiated by addition of Taq ligase under hot startconditions. The LDR probes ligate to their adjacent probes only in thepresence of target sequence which gives perfect complementarity at thejunction site. The products may be detected in two different formats. Inthe first format 4a, used in the prior art, fluorescently-labeled LDRprobes contain different length poly A or hexaethylene oxide tails.Thus, each LDR product, resulting from ligation to normal DNA with aslightly different mobility, yields a ladder of peaks. A germlinemutation would generate a new peak on the electrophorogram. The size ofthe new peak will approximate the amount of the mutation present in theoriginal sample; 0% for homozygous normal, 50% for heterozygous carrier,or 100% for homozygous mutant. In the second format 4b, in accordancewith the present invention, each allele-specific probe contains e.g., 24additional nucleotide bases on their 5′ ends. These sequences are uniqueaddressable sequences which will specifically hybridize to theircomplementary address sequences on an addressable array. In the LDRreaction, each allele-specific probe can ligate to its adjacentfluorescently labeled common probe in the presence of the correspondingtarget sequence. Wild type and mutant alleles are captured on adjacentaddresses on the array. Unreacted probes are washed away. The black dotsindicate 100% signal for the wild type allele. The white dots indicate0% signal for the mutant alleles. The shaded dots indicate the oneposition of germline mutation, 50% signal for each allele.

FIG. 2 depicts detection of somatic cell mutations in the p53 tumorsuppressor gene but is general for all low sensitivity mutationdetection. In step 1, DNA samples are prepared and exons 5-9 are PCRamplified as three fragments using fluorescent PCR primers. This allowsfor fluorescent quantification of PCR products in step 2 using capillaryor gel electrophoresis. In step 3, the products are spiked with a 1/100dilution of marker DNA (for each of the three fragments). This DNA ishomologous to wild type DNA, except it contains a mutation which is notobserved in cancer cells, but which may be readily detected with theappropriate LDR probes. The mixed DNA products in step 4 are diluted20-fold into buffer containing all the LDR probes which are specificonly to mutant or marker alleles. In step 5, the ligase reaction isinitiated by addition of Taq ligase under hot start conditions. The LDRprobes ligate to their adjacent probes only in the presence of targetsequences which give perfect complementarity at the junction site. Theproducts may be detected in the same two formats described in FIG. 1. Inthe format of step 6a, which is used in the prior art, products areseparated by capillary or gel electrophoresis, and fluorescent signalsare quantified. Ratios of mutant peaks to marker peaks give approximateamount of cancer mutations present in the original sample divided by100. In the format of step 6b, in accordance with the present invention,products are detected by specific hybridization to complementarysequences on an addressable array. Ratios of fluorescent signals inmutant dots to marker dots give the approximate amount of cancermutations present in the original sample divided by 100.

The ligase detection reaction process, in accordance with the presentinvention, is best understood by referring to FIGS. 3-10. It isdescribed generally in WO 90/17239 to Barany et al., F. Barany et al.,“Cloning, Overexpression and Nucleotide Sequence of a Thermostable DNALigase-encoding Gene,” Gene, 109:1-11 (1991), and F. Barany, “GeneticDisease Detection and DNA Amplification Using Cloned ThermostableLigase,” Proc. Natl. Acad. Sci. USA, 88:189-193 (1991), the disclosuresof which are hereby incorporated by reference. In accordance with thepresent invention, the ligase detection reaction can use 2 sets ofcomplementary oligonucleotides. This is known as the ligase chainreaction which is described in the 3 immediately preceding references,which are hereby incorporated by reference. Alternatively, the ligasedetection reaction can involve a single cycle which is known as theoligonucleotide ligation assay. See Landegren, et al., “ALigase-Mediated Gene Detection Technique,” Science 241:1077-80 (1988);Landegren, et al., “DNA Diagnostics—Molecular Techniques andAutomation,” Science 242:229-37 (1988); and U.S. Pat. No. 4,988,617 toLandegren, et al.

During the ligase detection reaction phase of the process, thedenaturation treatment is carried out at a temperature of 80-105° C.,while hybridization takes place at 50-85° C. Each cycle comprises adenaturation treatment and a thermal hybridization treatment which intotal is from about one to five minutes long. Typically, the ligationdetection reaction involves repeatedly denaturing and hybridizing for 2to 50 cycles. The total time for the ligase detection reaction phase ofthe process is 1 to 250 minutes.

The oligonucleotide probe sets can be in the form of ribonucleotides,deoxynucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, peptide nucleotide analogues, modified peptidenucleotide analogues, modified phosphate-sugar-backboneoligonucleotides, nucleotide analogs, and mixtures thereof.

In one variation, the oligonucleotides of the oligonucleotide probe setseach have a hybridization or melting temperature (i.e. T_(m)) of 66-70°C. These oligonucleotides are 20-28 nucleotides long.

It may be desirable to destroy chemically or enzymatically unconvertedLDR oligonucleotide probes that contain addressable nucleotidearray-specific portions prior to capture of the ligation products on aDNA array. Such unconverted probes will otherwise compete with ligationproducts for binding at the addresses on the array of the solid supportwhich contain complementary sequences. Destruction can be accomplishedby utilizing an exonuclease, such as exonuclease III (L-H Guo and R. Wu,Methods in Enzymology 100:60-96 (1985), which is hereby incorporated byreference) in combination with LDR probes that are blocked at the endsand not involved with ligation of probes to one another. The blockingmoiety could be a reporter group or a phosphorothioate group. T. T.Nikiforow, et al., “The Use of Phosphorothioate Primers and ExonucleaseHydrolysis for the Preparation of Single-stranded PCR Products and theirDetection by Solid-phase Hybridization,” PCR Methods and Applications,3:p. 285-291 (1994), which is hereby incorporated by reference. Afterthe LDR process, unligated probes are selectively destroyed byincubation of the reaction mixture with the exonuclease. The ligatedprobes are protected due to the elimination of free 3′ ends which arerequired for initiation of the exonuclease reaction. This approachresults in an increase in the signal-to-noise ratio, especially wherethe LDR reaction forms only a small amount of product. Since unligatedoligonucleotides compete for capture by the capture oligonucleotide,such competition with the ligated oligonucleotides lowers the signal. Anadditional advantage of this approach is that unhybridizedlabel-containing sequences are degraded and, therefore, are less able tocause a target-independent background signal, because they can beremoved more easily from the DNA array by washing.

The oligonucleotide probe sets, as noted above, have a reporter labelsuitable for detection. Useful labels include chromophores, fluorescentmoieties, enzymes, antigens, heavy metals, magnetic probes, dyes,phosphorescent groups, radioactive materials, chemiluminescent moieties,and electrochemical detecting moieties. The capture oligonucleotides canbe in the form of ribonucleotides, deoxyribonucleotides, modifiedribonucleotides, modified deoxyribonucleotides, peptide nucleotideanalogues, modified peptide nucleotide analogues, modifiedphosphate-sugar backbone oligonucleotides, nucleotide analogues, andmixtures thereof. Where the process of the present invention involvesuse of a plurality of oligonucleotide sets, the second oligonucleotideprobes can be the same, while the addressable array-specific portions ofthe first oligonucleotide probes differ. Alternatively, the addressablearray-specific portions of the first oligonucleotide probes may be thesame, while the reporter labels of the second oligonucleotide probes aredifferent.

Prior to the ligation detection reaction phase of the present invention,the sample is preferably amplified by an initial target nucleic acidamplification procedure. This increases the quantity of the targetnucleotide sequence in the sample. For example, the initial targetnucleic acid amplification may be accomplished using the polymerasechain reaction process, self-sustained sequence replication, or Q-βreplicase-mediated RNA amplification. The polymerase chain reactionprocess is the preferred amplification procedure and is fully describedin H. Erlich, et. al., “Recent Advances in the Polymerase ChainReaction,” Science 252: 1643-50 (1991); M. Innis, et. al., PCRProtocols: A Guide to Methods and Applications, Academic Press: New York(1990); and R. Saiki, et. al., “Primer-directed Enzymatic Amplificationof DNA with a Thermostable DNA Polymerase,” Science 239: 487-91 (1988),which are hereby incorporated by reference. J. Guatelli, et. al.,“Isothermal, in vitro Amplification of Nucleic Acids by a MultienzymeReaction Modeled After Retroviral Replication,” Proc. Natl. Acad. Sci.USA 87: 1874-78 (1990), which is hereby incorporated by reference,describes the self-sustained sequence replication process. The Q-βreplicase-mediated RNA amplification is disclosed in F. Kramer, et. al.,“Replicatable RNA Reporters,” Nature 339: 401-02 (1989), which is herebyincorporated by reference.

The use of the polymerase chain reaction process and then the ligasedetection process, in accordance with the present invention, is shown inFIG. 3. Here, homo- or heterozygosity at two polymorphisms (i.e. alleledifferences) are on the same gene. Such allele differences canalternatively be on different genes.

As shown in FIG. 3, the target nucleic acid, when present in the form ofa double stranded DNA molecule is denatured to separate the strands.This is achieved by heating to a temperature of 80-105° C. Polymerasechain reaction primers are then added and allowed to hybridize to thestrands, typically at a temperature of 20-85° C. A thermostablepolymerase (e.g., Thermus aquaticus polymerase) is also added, and thetemperature is then adjusted to 50-85° C. to extend the primer along thelength of the nucleic acid to which the primer is hybridized. After theextension phase of the polymerase chain reaction, the resulting doublestranded molecule is heated to a temperature of 80-105° C. to denaturethe molecule and to separate the strands. These hybridization,extension, and denaturation steps may be repeated a number of times toamplify the target to an appropriate level.

Once the polymerase chain reaction phase of the process is completed,the ligation detection reaction phase begins, as shown in FIG. 3. Afterdenaturation of the target nucleic acid, if present as a double strandedDNA molecule, at a temperature of 80-105° C., preferably 94° C.,ligation detection reaction oligonucleotide probes for one strand of thetarget nucleotide sequence are added along with a ligase (for example,as shown in FIG. 3, a thermostable ligase like Thermus aquaticusligase). The oligonucleotide probes are then allowed to hybridize to thetarget nucleic acid molecule and ligate together, typically, at atemperature of 45-85° C., preferably, 65° C. When there is perfectcomplementarity at the ligation junction, the oligonucleotides can beligated together. Where the variable nucleotide is T or A, the presenceof T in the target nucleotide sequence will cause the oligonucleotideprobe with the addressable array-specific portion Z1 to ligate to theoligonucleotide probe with the reporter label F, and the presence of Ain the target nucleotide sequence will cause the oligonucleotide probewith the addressable array-specific portion Z2 to ligate to theoligonucleotide probe with reporter label F. Similarly, where thevariable nucleotide is A or G, the presence of T in the targetnucleotide sequence will cause the oligonucleotide probe withaddressable array-specific portion Z4 to ligate to the oligonucleotideprobe with the reporter label F, and the presence of C in the targetnucleotide sequence will cause the oligonucleotide probe with theaddressable array-specific portion Z3 to ligate to the oligonucleotideprobe with reporter label F. Following ligation, the material is againsubjected to denaturation to separate the hybridized strands. Thehybridization/ligation and denaturation steps can be carried through oneor more cycles (e.g., 1 to 50 cycles) to amplify the target signal.Fluorescent ligation products (as well as unligated oligonucleotideprobes having an addressable array-specific portion) are captured byhybridization to capture probes complementary to portions Z1, Z2, Z3,and Z4 at particular addresses on the addressable arrays. The presenceof ligated oligonucleotides is then detected by virtue of the label Foriginally on one of the oligonucleotides. In FIG. 3, ligated productsequences hybridize to the array at addresses with captureoligonucleotides complementary to addressable array-specific portions Z1and Z3, while unligated oligonucleotide probes with addressablearray-specific portions Z2 and Z4 hybridize to their complementarycapture oligonucleotides. However, since only the ligated productsequences have label F, only their presence is detected.

FIG. 4 is a flow diagram of a PCR/LDR process, in accordance with thepresent invention, which distinguishes any possible base at a givensite. Appearance of fluorescent signal at the addresses complementary toaddressable array-specific portions Z1, Z2, Z3, and Z4 indicates thepresence of A, G, C, and T alleles in the target nucleotide sequence,respectively. Here, the presence of the A and C alleles in the targetnucleotide sequences is indicated due to the fluorescence at theaddresses on the solid support with capture oligonucleotide probescomplementary to portions Z1 and Z3, respectively. Note that in FIG. 4the addressable array-specific portions are on the discriminatingoligonucleotide probes, and the discriminating base is on the 3′ end ofthese probes.

FIG. 5 is a flow diagram of a PCR/LDR process, in accordance with thepresent invention, for detecting the presence of any possible base attwo nearby sites. Here, the LDR primers are able to overlap, yet arestill capable of ligating provided there is perfect complementarity atthe junction. This distinguishes LDR from other approaches, such asallele-specific PCR where overlapping primers would interfere with oneanother. In FIG. 5, the first nucleotide position is heterozygous at theA and C alleles, while the second nucleotide position is heterozygous tothe G, C, and T alleles. As in FIG. 4, the addressable array-specificportions are on the discriminating oligonucleotide probes, and thediscriminating base is on the 3′ end of these probes. The reporter group(e.g., the fluorescent label) is on the 3′ end of the commonoligonucleotide probes. This is possible for example with the 21hydroxylase gene, where each individual has 2 normal and 2 pseudogenes,and, at the intron 2 splice site (nucleotide 656), there are 3 possiblesingle bases (G, A, and C). Also, this can be used to detect lowabundance mutations in HIV infections which might indicate emergence ofdrug resistant (e.g., to AZT) strains. Returning to FIG. 5, appearanceof fluorescent signal at the addresses complementary to addressablearray-specific portions Z1, Z2, Z3, Z4, Z5, Z6, Z7, and Z8 indicates thepresence of the A, G, C, and T, respectively, in the site heterozygousat the A and C alleles, and A, G, C, and T, respectively, in the siteheterozygous at the G, C, and T alleles.

FIG. 6 is a flow diagram of a PCR/LDR process, in accordance with thepresent invention, where insertions (top left set of probes) anddeletions (bottom right set of probes) are distinguished. On the left,the normal sequence contains 5 A's in a polyA tract. The mutant sequencehas an additional 2As inserted into the tract. Therefore, the LDRproducts with addressable array-specific portions Z1 (representing thenormal sequence) and Z3 (representing a 2 base pair insertion) would befluorescently labeled by ligation to the common primer. While the LDRprocess (e.g., using a thermostable ligase enzyme) has no difficultydistinguishing single base insertions or deletions in mononucleotiderepeats, allele-specific PCR is unable to distinguish such differences,because the 3′ base remains the same for both alleles. On the right, thenormal sequence is a (CA)5 repeat (i.e. CACACACACA (SEQ ID NO: 1)). Themutant contains two less CA bases than the normal sequence (i.e.CACACA). These would be detected as fluorescent LDR products at theaddressable array-specific portions Z8 (representing the normalsequence) and Z6 (representing the 2 CA deletion) addresses. Theresistance of various infectious agents to drugs can also be determinedusing the present invention. In FIG. 6, the presence of ligated productsequences, as indicated by fluorescent label F, at the address havingcapture oligonucleotides complementary to Z1 and Z3 demonstrates thepresence of both the normal and mutant poly A sequences. Similarly, thepresence of ligated product sequences, as indicated by fluorescent labelF, at the address having capture oligonucleotides complementary to Z6and Z8 demonstrates the presence of both the normal CA repeat and asequence with one repeat unit deleted.

FIG. 7 is a flow diagram of a PCR/LDR process, in accordance with thepresent invention, using addressable array-specific portions to detect alow abundance mutation in the presence of an excess of normal sequence.FIG. 7 shows codon 12 of the K-ras gene, sequence GGT, which codes forglycine (“Gly”). A small percentage of the cells contain the G to Amutation in GAT, which codes for aspartic acid (“Asp”). The LDR probesfor wild-type (i.e. normal sequences) are missing from the reaction. Ifthe normal LDR probes (with the discriminating base=G) were included,they would ligate to the common probes and overwhelm any signal comingfrom the mutant target. Instead, as shown in FIG. 7, the existence of aligated product sequence with fluorescent label F at the address with acapture oligonucleotide complementary to addressable array-specificportion Z4 indicates the presence of the aspartic acid encoding mutant.

FIG. 8 is a flow diagram of a PCR/LDR process, in accordance with thepresent invention, where the addressable array-specific portion isplaced on the common oligonucleotide probe, while the discriminatingoligonucleotide probe has the reporter label. Allele differences aredistinguished by different fluorescent signals, F1, F2, F3, and F4. Thismode allows for a more dense use of the arrays, because each position ispredicted to light up with some group. It has the disadvantage ofrequiring fluorescent groups which have minimal overlap in theiremission spectra and will require multiple scans. It is not ideallysuitable for detection of low abundance alleles (e.g., cancer associatedmutations).

FIG. 9 is a flow diagram of a PCR/LDR process, in accordance with thepresent invention, where both adjacent and nearby alleles are detected.The adjacent mutations are right next to each other, and one set ofoligonucleotide probes discriminates the bases on the 3′ end of thejunction (by use of different addressable array-specific portions Z1,Z2, Z3, and Z4), while the other set of oligonucleotide probesdiscriminates the bases on the 5′ end of the junction (by use ofdifferent fluorescent reporter labels F1, F2, F3, and F4). In FIG. 9,codons in a disease gene (e.g. CFTR for cystic fibrosis) encoding Glyand arginine (“Arg”), respectively, are candidates for germlinemutations. The detection results in FIG. 9 show the Gly (GGA; indicatedby the ligated product sequence having portion Z2 and label F2) has beenmutated to glutamic acid (“Glu”) (GAA; indicated by the ligated productsequence having portion Z2 and label F1), and the Arg (CGG; indicated bythe ligated product sequence having portion Z7 and label F2) has beenmutated to tryptophan (“Trp”) (TGG; indicated by the ligated productsequence with portion Z8 and label F2). Therefore, the patient is acompound heterozygous individual (i.e. with allele mutations in bothgenes) and will have the disease.

FIG. 10 is a flow diagram of a PCR/LDR process, in accordance with thepresent invention, where all possible single-base mutations for a singlecodon are detected. Most amino acid codons have a degeneracy in thethird base, thus the first two positions can determine all the possiblemutations at the protein level. These amino acids include arginine,leucine, serine, threonine, proline, alanine, glycine, and valine.However, some amino acids are determined by all three bases in the codonand, thus, require the oligonucleotide probes to distinguish mutationsin 3 adjacent positions. By designing four oligonucleotide probescontaining the four possible bases in the penultimate position to the 3′end, as well as designing an additional four capture oligonucleotidescontaining the four possible bases at the 3′ end, as shown in FIG. 10,this problem has been solved. The common oligonucleotide probes with thereporter labels only have two fluorescent groups which correspond to thecodon degeneracies and distinguish between different ligated productsequences which are captured at the same array address. For example, asshown in FIG. 10, the presence of a glutamine (“Gln”) encoding codon(i.e., CAA and CAG) is indicated by the presence of a ligated productsequence containing portion Z1 and label F2. Likewise, the existence ofa Gln to histidine (“His”) encoding mutation (coded by the codon CAC) isindicated by the presence of ligated product sequences with portion Z1and label F2 and with portion Z7 and label F2 There is an internalredundancy built into this assay due to the fact that primers Z1 and Z7have the identical sequence.

A particularly important aspect of the present invention is itscapability to quantify the amount of target nucleotide sequence in asample. This can be achieved in a number of ways by establishingstandards which can be internal (i.e. where the standard establishingmaterial is amplified and detected with the sample) or external (i.e.where the standard establishing material is not amplified, and isdetected with the sample).

In accordance with one quantification method, the signal generated bythe reporter label, resulting from capture of ligated product sequencesproduced from the sample being analyzed, are detected. The strength ofthis signal is compared to a calibration curve produced from signalsgenerated by capture of ligated product sequences in samples with knownamounts of target nucleotide sequence. As a result, the amount of targetnucleotide sequence in the sample being analyzed can be determined. Thistechniques involves use of an external standard.

Another quantification method, in accordance with the present invention,relates to an internal standard. Here, a known amount of one or moremarker target nucleotide sequences are added to the sample. In addition,a plurality of marker-specific oligonucleotide probe sets are addedalong with the ligase, the previously-discussed oligonucleotide probesets, and the sample to a mixture. The marker-specific oligonucleotideprobe sets have (1) a first oligonucleotide probe with a target-specificportion complementary to the marker target nucleotide sequence and anaddressable array-specific portion complementary to captureoligonucleotides on the solid support and (2) a second oligonucleotideprobe with a target-specific portion complementary to the marker targetnucleotide sequence and a detectable reporter label. The oligonucleotideprobes in a particular marker-specific oligonucleotide set are suitablefor ligation together when hybridized adjacent to one another on acorresponding marker target nucleotide sequence. However, there is amismatch which interferes with such ligation when hybridized to anyother nucleotide sequence present in the sample or added markersequences. The presence of ligated product sequences captured on thesolid support is identified by detection of reporter labels. The amountof target nucleotide sequences in the sample is then determined bycomparing the amount of captured ligated product generated from knownamounts of marker target nucleotide sequences with the amount of otherligated product sequences captured.

Another quantification method in accordance with the present inventioninvolves analysis of a sample containing two or more of a plurality oftarget nucleotide sequences with a plurality of sequence differences.Here, ligated product sequences corresponding to the target nucleotidesequences are detected and distinguished by any of thepreviously-discussed techniques. The relative amounts of the targetnucleotide sequences in the sample are then quantified by comparing therelative amounts of captured ligated product sequences generated. Thisprovides a quantitative measure of the relative level of the targetnucleotide sequences in the sample.

The ligase detection reaction process phase of the present invention canbe preceded by the ligase chain reaction process to achieveoligonucleotide product amplification. This process is fully describedin F. Barany, et. al., “Cloning, Overexpression and Nucleotide Sequenceof a Thermostable DNA Ligase-encoding Gene,” Gene 109: 1-11 (1991) andF. Barany, “Genetic Disease Detection and DNA Amplification Using ClonedThermostable Ligase,” Proc. Natl. Acad. Sci. USA 88: 189-93 (1991),which are hereby incorporated by reference. Instead of using the ligasechain reaction to achieve amplification, a transcription-basedamplifying procedure can be used.

The preferred thermostable ligase is that derived from Thermusaquaticus. This enzyme can be isolated from that organism. M. Takahashi,et al., “Thermophillic DNA Ligase,” J. Biol. Chem. 259:10041-47 (1984),which is hereby incorporated by reference. Alternatively, it can beprepared recombinantly. Procedures for such isolation as well as therecombinant production of Thermus aquaticus ligase as well as Thermusthemophilus ligase) are disclosed in WO 90/17239 to Barany, et. al., andF. Barany, et al., “Cloning, Overexpression and Nucleotide Sequence of aThermostable DNA-Ligase Encoding Gene,” Gene 109:1-11 (1991), which arehereby incorporated by reference. These references contain completesequence information for this ligase as well as the encoding DNA. Othersuitable ligases include E. coli ligase, T4 ligase, and Pyrococcusligase.

The ligation amplification mixture may include a carrier DNA, such assalmon sperm DNA.

The hybridization step, which is preferably a thermal hybridizationtreatment, discriminates between nucleotide sequences based on adistinguishing nucleotide at the ligation junctions. The differencebetween the target nucleotide sequences can be, for example, a singlenucleic acid base difference, a nucleic acid deletion, a nucleic acidinsertion, or rearrangement. Such sequence differences involving morethan one base can also be detected. Preferably, the oligonucleotideprobe sets have substantially the same length so that they hybridize totarget nucleotide sequences at substantially similar hybridizationconditions. As a result, the process of the present invention is able todetect infectious diseases, genetic diseases, and cancer. It is alsouseful in environmental monitoring, forensics, and food science.

A wide variety of infectious diseases can be detected by the process ofthe present invention. Typically, these are caused by bacterial, viral,parasite, and fungal infectious agents. The resistance of variousinfectious agents to drugs can also be determined using the presentinvention.

Bacterial infectious agents which can be detected by the presentinvention include Escherichia coli, Salmonella, Shigella, Klebsiella,Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis,Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasteurella,Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcusaureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria,Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea,Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis,Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponemapalladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsialpathogens, Nocardia, and Acitnomycetes.

Fungal infectious agents which can be detected by the present inventioninclude Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasmacapsulatum, Coccidioides immitis, Paracoccicioides brasiliensis, Candidaalbicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrixschenckii, Chromomycosis, and Maduromycosis.

Viral infectious agents which can be detected by the present inventioninclude human immunodeficiency virus, human T-cell lymphocytotrophicvirus, hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis CVirus), Epstein-Barr Virus, cytomegalovirus, human papillomaviruses,orthomyxo viruses, paramyxo viruses, adenoviruses, corona-viruses,rhabdo-viruses, polio viruses, toga viruses, bunya viruses, arenaviruses, rubella viruses, and reo viruses.

Parasitic agents which can be detected by the present invention includePlasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodiumovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosomaspp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonasspp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobiusvermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculusmedinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystiscarinii, and Necator americanis.

The present invention is also useful for detection of drug resistance byinfectious agents. For example, vancomycin-resistant Enterococcusfaecium, methicillin-resistant Staphylococcus aureus,penicillin-resistant Streptococcus pneumoniae, multi-drug resistantMycobacterium tuberculosis, and AZT-resistant human immunodeficiencyvirus can all be identified with the present invention.

Genetic diseases can also be detected by the process of the presentinvention. This can be carried out by prenatal screening for chromosomaland genetic aberrations or post natal screening for genetic diseases.Examples of detectable genetic diseases include: 21 hydroxylasedeficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome,Duchenne Muscular Dystrophy, Down Syndrome or other trisomies, heartdisease, single gene diseases, HLA typing, phenylketonuria, sickle cellanemia, Tay-Sachs Syndrome, thalassemia, Klinefelter's Syndrome,Huntington's Disease, autoimmune diseases, lipidosis, obesity defects,hemophilia, inborn errors in metabolism, and diabetes.

Cancers which can be detected by the process of the present inventiongenerally involve oncogenes, tumor suppressor genes, or genes involvedin DNA amplification, replication, recombination, or repair. Examples ofthese include: BRCA1 gene, p53 gene, Familial polyposis coli, Her2/Neuamplification, Bcr/Ab1, K-ras gene, human papillomavirus Types 16 and18, leukemia, colon cancer, breast cancer, lung cancer, prostate cancer,brain tumors, central nervous system tumors, bladder tumors, melanomas,liver cancer, osteosarcoma and other bone cancers, testicular andovarian carcinomas, ENT tumors, and loss of heterozygosity.

In the area of environmental monitoring, the present invention can beused for detection, identification, and monitoring of pathogenic andindigenous microorganisms in natural and engineered ecosystems andmicrocosms such as in municipal waste water purification systems andwater reservoirs or in polluted areas undergoing bioremediation. It isalso possible to detect plasmids containing genes that can metabolizexenobiotics, to monitor specific target microorganisms in populationdynamic studies, or either to detect, identify, or monitor geneticallymodified microorganisms in the environment and in industrial plants.

The present invention can also be used in a variety or forensic areas,including for human identification for military personnel and criminalinvestigation, paternity testing and family relation analysis, HLAcompatibility typing, and screening blood, sperm, or transplantationorgans for contamination.

In the food and feed industry, the present invention has a wide varietyof applications. For example, it can be used for identification andcharacterization of production organisms such as yeast for production ofbeer, wine, cheese, yogurt, bread, etc. Another area of use is withregard to quality control and certification of products and processes(e.g., livestock, pasteurization, and meat processing) for contaminants.Other uses include the characterization of plants, bulbs, and seeds forbreeding purposes, identification of the presence of plant-specificpathogens, and detection and identification of veterinary infections.

Desirably, the oligonucleotide probes are suitable for ligation togetherat a ligation junction when hybridized adjacent to one another on acorresponding target nucleotide sequence due to perfect complementarityat the ligation junction. However, when the oligonucleotide probes inthe set are hybridized to any other nucleotide sequence present in thesample, there is a mismatch at a base at the ligation junction whichinterferes with ligation. Most preferably, the mismatch is at the baseadjacent the 3′ base at the ligation junction. Alternatively, themismatch can be at the bases adjacent to bases at the ligation junction.

The process of the present invention is able to detect the first andsecond nucleotide sequences in the sample in an amount of 100 attomolesto 250 femtomoles. By coupling the LDR step with a primarypolymerase-directed amplification step, the entire process of thepresent invention is able to detect target nucleotide sequences in asample containing as few as a single molecule. Furthermore, PCRamplified products, which often are in the picomole amounts, may easilybe diluted within the above range. The ligase detection reactionachieves a rate of formation of mismatched ligated product sequenceswhich is less than 0.005 of the rate of formation of matched ligatedproduct sequences.

Once the ligation phase of the process is completed, the capture phaseis initiated. During the capture phase of the process, the mixture iscontacted with the solid support at a temperature of 45-90° C. and for atime period of up to 60 minutes. Hybridizations may be accelerated byadding volume exclusion or chaotropic agents. When an array consists ofdozens to hundreds of addresses, it is important that the correctligation products have an opportunity to hybridize to the appropriateaddress. This may be achieved by the thermal motion of oligonucleotidesat the high temperatures used, by mechanical movement of the fluid incontact with the array surface, or by moving the oligonucleotides acrossthe array by electric fields. After hybridization, the array is washedsequentially with a low stringency wash buffer and then a highstringency wash buffer.

It is important to select capture oligonucleotides and addressablenucleotide sequences which will hybridize in a stable fashion. Thisrequires that the oligonucleotide sets and the capture oligonucleotidesbe configured so that the oligonucleotide sets hybridize to the targetnucleotide sequences at a temperature less than that which the captureoligonucleotides hybridize to the addressable array-specific portions.Unless the oligonucleotides are designed in this fashion, false positivesignals may result due to capture of adjacent unreacted oligonucleotidesfrom the same oligonucleotide set which are hybridized to the target.

The detection phase of the process involves scanning and identifying ifligation of particular oligonucleotide sets occurred and correlatingligation to a presence or absence of the target nucleotide sequence inthe test sample. Scanning can be carried out by scanning electronmicroscopy, confocal microscopy, charge-coupled device, scanningtunneling electron microscopy, infrared microscopy, atomic forcemicroscopy, electrical conductance, and fluorescent or phosphor imaging.Correlating is carried out with a computer.

Another-aspect of the present invention relates to a method of formingan array of oligonucleotides on a solid support. This method involvesproviding a solid support having an array of positions each suitable forattachment of an oligonucleotide. A linker or support (preferablynon-hydrolyzable), suitable for coupling an oligonucleotide to the solidsupport at each of the array positions, is attached to the solidsupport. An array of oligonucleotides on a solid support is formed by aseries of cycles of activating selected array positions for attachmentof multimer nucleotides and attaching multimer nucleotides at theactivated array positions.

Yet another aspect of the present invention relates to an array ofoligonucleotides on a solid support per se. The solid support has anarray of positions each suitable for an attachment of anoligonucleotide. A linker or support (preferably non-hydrolyzable),suitable for coupling an oligonucleotide to the solid support, isattached to the solid support at each of the array positions. An arrayof oligonucleotides are placed on a solid support with at least some ofthe array positions being occupied by oligonucleotides having greaterthan sixteen nucleotides.

In the method of forming arrays, multimer oligonucleotides fromdifferent multimer oligonucleotide sets are attached at different arraypositions on a solid support. As a result, the solid support has anarray of positions with different groups of multimer oligonucleotidesattached at different positions.

The 1,000 different addresses can be unique capture oligonucleotidesequences (e.g., 24-mer) linked covalently to the target-specificsequence (e.g., approximately 20- to 25-mer) of a LDR oligonucleotideprobe. A capture oligonucleotide probe sequence does not have anyhomology to either the target sequence or to other sequences on genomeswhich may be present in the sample. This oligonucleotide probe is thencaptured by its addressable array-specific portion, a sequencecomplementary to the capture oligonucleotide on the addressable solidsupport array. The concept is shown in two possible formats, forexample, for detection of the p53 R248 mutation (FIGS. 13A-C).

In FIGS. 13A-C, two alternative formats for oligonucleotide probe designto identify the presence of a germ line mutation in codon 248 of the p53tumor suppressor gene are shown. The wild type sequence codes forarginine (R248), while the cancer mutation codes for tryptophan (R248W).FIGS. 13B and 13C are schematic diagrams of the captureoligonucleotides. The thick horizontal line depicts the membrane orsolid surface containing the addressable array. The thin curved linesindicate a flexible linker arm. The thicker lines indicate a captureoligonucleotide sequence, attached to the solid surface in the C to Ndirection. For illustrative purposes, the capture oligonucleotides aredrawn vertically, making the linker arm in section B appear “stretched”.Since the arm is flexible, the capture oligonucleotide will be able tohybridize 5′ to C and 3′ to N in each case, as dictated by base paircomplementarity. A similar orientation of oligonucleotide hybridizationwould be allowed if the oligonucleotides were attached to the membraneat the N-terminus. In this case, DNA/PNA hybridization would be instandard antiparallel 5′ to 3′ and 3′ to 5′. Other modifiedsugar-phosphate backbones would be used in a similar fashion. The topportion of FIG. 13A shows two LDR primers that are designed todiscriminate wild type and mutant p53 by containing the discriminatingbase C or T at the 3′ end. In the presence of the correct target DNA andTth ligase, the discriminating probe is covalently attached to a commondownstream oligonucleotide. The downstream oligonucleotide isfluorescently labeled. The discriminating oligonucleotides aredistinguished by the presence of unique addressable array-specificportions, Z1 and Z2, at each of their 5′ ends. A black dot indicatesthat target dependent ligation has taken place. After ligation,oligonucleotide probes may be captured by their complementaryaddressable array-specific portions at unique addresses on the array asshown in FIG. 13B. Both ligated and unreacted oligonucleotide probes arecaptured by the oligonucleotide array. Unreacted fluorescently labeledcommon primers and target DNA are then washed away at a high temperature(approximately 65° C. to 80° C.) and low salt. Mutant signal isdistinguished by detection of fluorescent signal at the captureoligonucleotide complementary to addressable array-specific portion Z1,while wild type signal appears at the capture oligonucleotidecomplementary to addressable array-specific portion Z2. Heterozygosityis indicated by equal signals at the capture oligonucleotidescomplementary to addressable array-specific portions Z1 and Z2. Thesignals may be quantified using a fluorescent imager. This format uses aunique address for each allele and may be preferred for achieving veryaccurate detection of low levels of signal (30 to 100 attomoles of LDRproduct). FIG. 13C shows the discriminating signals may be quantifiedusing a fluorescent imager. This format uses a unique address whereoligonucleotide probes are distinguished by having different fluorescentgroups, F1 and F2, on their 5′ end. Either oligonucleotide probe may beligated to a common downstream oligonucleotide probe containing anaddressable array-specific portion Z1 on its 3′ end. In this format,both wild type and mutant LDR products are captured at the same addresson the array, and are distinguished by their different fluorescence.This format allows for a more efficient use of the array and may bepreferred when trying to detect hundreds of potential germlinemutations.

The solid support can be made from a wide variety of materials. Thesubstrate may be biological, nonbiological, organic, inorganic, or acombination of any of these, existing as particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, slides, discs, membranes, etc. Thesubstrate may have any convenient shape, such as a disc, square, circle,etc. The substrate is preferably flat but may take on a variety ofalternative surface configurations. For example, the substrate maycontain raised or depressed regions on which the synthesis takes place.The substrate and its surface preferably form a rigid support on whichto carry out the reactions described herein. The substrate and itssurface is also chosen to provide appropriate light-absorbingcharacteristics. For instance, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SiN₄, modified silicon, or any one of a wide variety of gels or polymerssuch as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,polystyrene, polycarbonate, polyethylene, polypropylene, polyvinylchloride, poly(methyl acrylate), poly(methyl methacrylate), orcombinations thereof. Other substrate materials will be readily apparentto those of ordinary skill in the art upon review of this disclosure. Ina preferred embodiment, the substrate is flat glass or single-crystalsilicon.

According to some embodiments, the surface of the substrate is etchedusing well known techniques to provide for desired surface features. Forexample, by way of the formation of trenches, v-grooves, mesastructures, raised platforms, or the like, the synthesis regions may bemore closely placed within the focus point of impinging light, beprovided with reflective “mirror” structures for maximization of lightcollection from fluorescent sources, or the like.

Surfaces on the solid substrate will usually, though not always, becomposed of the same material as the substrate. Thus, the surface may becomposed of any of a wide variety of materials, for example, polymers,plastics, ceramics, polysaccharides, silica or silica-based materials,carbon, metals, inorganic glasses, membranes, or composites thereof. Thesurface is functionalized with binding members which are attached firmlyto the surface of the substrate. Preferably, the surface functionalitieswill be reactive groups such as silanol, olefin, amino, hydroxyl,aldehyde, keto, halo, acyl halide, or carboxyl groups. In some cases,such functionalities preexist on the substrate. For example, silicabased materials have silanol groups, polysaccharides have hydroxylgroups, and synthetic polymers can contain a broad range of functionalgroups, depending on which monomers they are produced from.Alternatively, if the substrate does not contain the desired functionalgroups, such groups can be coupled onto the substrate in one or moresteps.

A variety of commercially-available materials, which include suitablymodified glass, plastic, or carbohydrate surfaces or a variety ofmembranes, can be used. Depending on the material, surface functionalgroups (e.g., silanol, hydroxyl, carboxyl, amino) may be present fromthe outset (perhaps as part of the coating polymer), or will require aseparate procedure (e.g., plasma amination, chromic acid oxidation,treatment with a functionalized side chain alkyltrichlorosilane) forintroduction of the functional group. Hydroxyl groups becomeincorporated into stable carbamate (urethane) linkages by severalmethods. Amino functions can be acylated directly, whereas carboxylgroups are activated, e.g., with N,N′-carbonyldiimidazole orwater-soluble carbodiimides, and reacted with an amino-functionalizedcompound. As shown in FIG. 11, the solid supports can be membranes orsurfaces with a starting functional group X. Functional grouptransformations can be carried out in a variety of ways (as needed) toprovide group X* which represents one partner in the covalent linkagewith group Y*. FIG. 11 shows specifically the grafting of PEG (i.e.polyethylene glycol), but the same repertoire of reactions can be used(however needed) to attach carbohydrates (with hydroxyl), linkers (withcarboxyl) and/or oligonucleotides that have been extended by suitablefunctional groups (amino or carboxyl). In some cases, group X* or Y* ispre-activated (isolatable species from a separate reaction);alternatively, activation occurs in situ. Referring to PEG as drawn inFIG. 11, Y and Y* can be the same (homobifunctional) or different(heterobifunctional); in the latter case, Y can be protected for furthercontrol of the chemistry. Unreacted amino groups will be blocked byacetylation or succinylation, to ensure a neutral or negatively chargedenvironment that “repels” excess unhybridized DNA. Loading levels can bedetermined by standard analytical methods. Fields, et al., “Principlesand Practice of Solid-Phase Peptide Synthesis,” Synthetic Peptides: AUser's Guide, G. Grant, Editor, W.H. Freeman and Co.: New York. p.77-183 (1992), which is hereby incorporated by reference.

One approach to applying functional groups on a silica-based supportsurface is to silanize with a molecule either having the desiredfunctional group (e.g., olefin, amino, hydroxyl, aldehyde, keto, halo,acyl halide, or carboxyl) or a molecule A able to be coupled to anothermolecule B containing the desired functional group. In the former case,functionalizing of glass- or silica-based solid supports with, forexample, an amino group is carried out by reacting with an aminecompound such as 3-aminopropyl triethoxysilane,3-aminopropylmethyldiethoxysilane, 3-aminopropyl dimethylethoxysilane,3-aminopropyl trimethoxysilane, N-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane, N-(2-aminoethyl-3-aminopropyl)trimethoxysilane,aminophenyl trimethoxysilane, 4-aminobutyldimethyl methoxysilane,4-aminobutyl triethoxysilane, aminoethylaminomethyphenethyltrimethoxysilane, or mixtures thereof. In the latter case, molecule Apreferably contains olefinic groups, such as vinyl, acrylate,methacrylate, or allyl, while molecule B contains olefinic groups andthe desired functional groups. In this case, molecules A and B arepolymerized together. In some cases, it is desirable to modify thesilanized surface to modify its properties (e.g., to impartbiocompatibility and to increase mechanical stability). This can beachieved by addition of olefinic molecule C along with molecule B toproduce a polymer network containing molecules A, B, and C.

Molecule A is defined by the following formula:

-   -   R¹ is H or CH₃    -   R² is (C═O)—O—R⁶, aliphatic group with or without functional        substituent(s), an aromatic group with or without functional        substituent(s), or mixed aliphatic/aromatic groups with or        without functional substituent(s);    -   R³ is an O-alkyl, alkyl, or halogen group;    -   R⁴ is an O-alkyl, alkyl, or halogen group;    -   R⁵ is an O-alkyl, alkyl, or halogen group; and    -   R⁶ is an aliphatic group with or without functional        substituent(s), an aromatic group with or without functional        substituent(s), or mixed aliphatic/aromatic groups with or        without functional substituent(s). Examples of Molecule A        include 3-(trimethoxysilyl)propyl methacrylate,        N-[3-(trimethoxysilyl)propyl]-N′-(4-vinylbenzyl)ethylenediamine,        triethoxyvinylsilane, triethylvinylsilane, vinyltrichlorosilane,        vinyltrimethoxysilane, and vinylytrimethylsilane.

Molecule B can be any monomer containing one or more of the functionalgroups described above. Molecule B is defined by the following formula:

-   -   (i)        -   R¹ is H or CH₃,        -   R² is (C═O), and        -   R³ is OH or Cl.    -   or    -   (ii)        -   R¹ is H or CH₃ and        -   R² is (C═O)—O—R⁴, an aliphatic group with or without            functional substituent(s), an aromatic group with or without            functional substituent(s), and mixed aliphatic/aromatic            groups with or without functional substituent(s); and        -   R³ is a functional group, such as OH, COOH, NH₂, halogen,            SH, COCl, or active ester; and        -   R⁴ is an aliphatic group with or without functional            substituent(s), an aromatic group with or without functional            substituent(s), or mixed aliphatic/aromatic groups with or            without functional substituent(s). Examples of molecule B            include acrylic acid, acrylamide, methacrylic acid,            vinylacetic acid, 4-vinylbenzoic acid, itaconic acid, allyl            amine, allylethylamine, 4-aminostyrene, 2-aminoethyl            methacrylate, acryloyl chloride, methacryloyl chloride,            chlorostyrene, dichlorostyrene, 4-hydroxystyrene,            hydroxymethyl styrene, vinylbenzyl alcohol, allyl alcohol,            2-hydroxyethyl methacrylate, or poly(ethylene            glycol)methacrylate.

Molecule C can be any molecule capable of polymerizing to molecule A,molecule B, or both and may optionally contain one or more of thefunctional groups described above. Molecule C can be any monomer orcross-linker, such as acrylic acid, methacrylic acid, vinylacetic acid,4-vinylbenzoic acid, itaconic acid, allyl amine, allylethylamine,4-aminostyrene, 2-aminoethyl methacrylate, acryloyl chloride,methacryloyl chloride, chlorostyrene, dichlorostyrene, 4-hydroxystyrene,hydroxymethyl styrene, vinylbenzyl alcohol, allyl alcohol,2-hydroxyethyl methacrylate, poly(ethylene glycol)methacrylate, methylacrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate,styrene, 1-vinylimidazole, 2-vinylpyridine, 4-vinylpyridine,divinylbenzene, ethylene glycol dimethacryarylate,N,N′-methylenediacrylamide, N,N′-phenylenediacrylamide,3,5-bis(acryloylamido)benzoic acid, pentaerythritol triacrylate,trimethylolpropane trimethacrylate, pentaerythritol tetraacrylate,trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate,trimethyolpropane ethoxylate (7/3 EO/OH) triacrylate, triethylolpropanepropoxylate (1 PO/OH) triacrylate, or trimethyolpropane propoxylate (2PO/PH triacrylate).

Generally, the functional-groups serve as starting points foroligonucleotides that will ultimately be coupled to the support. Thesefunctional groups can be reactive with an organic group that is to beattached to the solid support or it can be modified to be reactive withthat group, as through the use of linkers or handles. The functionalgroups can also impart various desired properties to the support.

After functionalization (if necessary) of the solid support, tailor-madepolymer networks containing activated functional groups that may serveas carrier sites for complementary oligonucleotide capture probes can begrafted to the support. The advantage of this approach is that theloading capacity of capture probes can thus be increased significantly,while physical properties of the intermediate solid-to-liquid phase canbe controlled better. Parameters that are subject to optimizationinclude the type and concentration of functional group-containingmonomers, as well as the type and relative concentration of thecrosslinkers that are used.

The surface of the functionalized substrate is preferably provided witha layer of linker molecules, although it will be understood that thelinker molecules are not required elements of the invention. The linkermolecules are preferably of sufficient length to permit polymers in acompleted substrate to interact freely with molecules exposed to thesubstrate. The linker molecules should be 6-50 atoms long to providesufficient exposure. The linker molecules may be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units,diamines, diacids, amino acids, or combinations thereof.

According to alternative embodiments, the linker molecules are selectedbased upon their hydrophilic/hydrophobic properties to improvepresentation of synthesized polymers to certain receptors. For example,in the case of a hydrophilic receptor, hydrophilic linker molecules willbe preferred to permit the receptor to approach more closely thesynthesized polymer.

According to another alternative embodiment, linker molecules are alsoprovided with a photocleavable group at any intermediate position. Thephotocleavable group is preferably cleavable at a wavelength differentfrom the protective group. This enables removal of the various polymersfollowing completion of the syntheses by way of exposure to thedifferent wavelengths of light.

The linker molecules can be attached to the substrate via carbon-carbonbonds using, for example, (poly)tri-fluorochloroethylene surfaces or,preferably, by siloxane bonds (using, for example, glass or siliconoxide surfaces). Siloxane bonds with the surface of the substrate may beformed in one embodiment via reactions of linker molecules bearingtrichlorosilyl groups. The linker molecules may optionally be attachedin an ordered array, i.e., as parts of the head groups in a polymerizedmonolayer. In alternative embodiments, the linker molecules are adsorbedto the surface of the substrate.

It is often desirable to introduce a PEG spacer with complementaryfunctionalization, prior to attachment of the starting linker for DNA orPNA synthesis. G. Barany, et al., “Novel Polyethylene Glycol-polystyrene(PEG-PS) Graft Supports for Solid-phase Peptide Synthesis,” ed. C. H.Schneider and A. N. Eberle., Leiden, The Netherlands: Escom SciencePublishers. 267-268 (1993); Zalipsky, et al., “Preparation andApplications of Polyethylene Glycol-polystyrene Graft Resin Supports forSolid-phase Peptide Synthesis,” Reactive Polymers, 22:243-58 (1994); J.M. Harris, ed. “Poly(Ethylene Glycol) Chemistry: Biotechnical andBiomedical Applications,” (1992), Plenum Press: New York, which arehereby incorporated by reference. Similarly, dextran layers can beintroduced as needed. Cass, et al., “Pilot, A New Peptide LeadOptimization Technique and Its Application as a General Library Method,in Peptides—Chemistry, Structure and Biology: Proceedings of theThirteenth American Peptide Symposium”, R. S. Hodges and J. A. Smith,Editor. (1994), Escom: Leiden, The Netherlands; Lofas, et al., “A NovelHydrogel Matrix on Gold Surface Plasma Resonance Sensors for Fast andEfficient Covalent Immobilization of Ligands,” J. Chem. Soc., Chem.Commun., pp. 1526-1528 (1990), which are hereby incorporated byreference. Particularly preferred linkers are tris(alkoxy)benzylcarbonium ions with dilute acid due to their efficient and specifictrapping with indole moieties. DNA oligonucleotides can be synthesizedand terminated with a residue of the amino acid tryptophan, andconjugated efficiently to supports that have been modified bytris(alkoxy)benzyl ester (hypersensitive acid labile (“HAL”)) ortris(alkoxy)benzylamide (“PAL”) linkers [F. Albericio, et al., J. Org.Chem., 55:3730-3743 (1990); F. Albericio and G. Barany, TetrahedronLett., 32:1015-1018 (1991)], which are hereby incorporated byreference). Other potentially rapid chemistries involve reaction ofthiols with bromoacetyl or maleimido functions. In one variation, theterminus of amino functionalized DNA is modified by bromoaceticanhydride, and the bromoacetyl function is captured by readilyestablished thiol groups on the support. Alternatively, an N-acetyl,S-tritylcysteine residue coupled to the end of the probe provides, aftercleavage and deprotection, a free thiol which can be captured by amaleimido group on the support. As shown in FIG. 12, chemicallysynthesized probes can be extended, on either end. Further variations ofthe proposed chemistries are readily envisaged. FIG. 12A shows that anamino group on the probe is modified by bromoacetic anhydride; thebromoacetyl function is captured by a thiol group on the support. FIG.12B shows that an N-acetyl, S-tritylcysteine residue coupled to the endof the probe provides, after cleavage and deprotection, a free thiolwhich is captured by a maleimido group on the support. FIG. 12C shows aprobe containing an oligo-tryptophanyl tail (n=1 to 3), which iscaptured after treatment of a HAL-modified solid support with diluteacid.

To prepare the arrays of the present invention, the solid supports mustbe charged with DNA oligonucleotides or PNA oligomers. This is achievedeither by attachment of pre-synthesized probes, or by direct assemblyand side-chain deprotection (without release of the oligomer) onto thesupport. Further, the support environment needs to be such as to allowefficient hybridization. Toward this end, two factors may be identified:(i) sufficient hydrophilic character of support material (e.g., PEG orcarbohydrate moieties) and (ii) flexible linker arms (e.g., hexaethyleneoxide or longer PEG chains) separating the probe from the supportbackbone. It should be kept in mind that numerous ostensibly “flatsurfaces” are quite thick at the molecular level. Lastly, it isimportant that the support material not provide significant backgroundsignal due to non-specific binding or intrinsic fluorescence.

The linker molecules and monomers used herein are provided with afunctional group to which is bound a protective group. Preferably, theprotective group is on the distal or terminal end of the linker moleculeopposite the substrate. The protective group may be either a negativeprotective group (i.e., the protective group renders the linkermolecules less reactive with a monomer upon exposure) or a positiveprotective group (i.e., the protective group renders the linkermolecules more reactive with a monomer upon exposure). In the case ofnegative protective groups, an additional step of reactivation will berequired. In some embodiments, this will be done by heating.

The protective group on the linker molecules may be selected from a widevariety of positive light-reactive groups preferably including nitroaromatic compounds such as o-nitrobenzyl derivatives or benzylsulfonyl.In a preferred embodiment, 6-nitroveratryloxycarbonyl (“NVOC”),2-nitrobenzyloxycarbonyl (“NBOC”), Benzyloxycarbonyl (“BOC”),fluorenylmethoxycarbonyl (“FMOC”), orα,α-dimethyl-dimethoxybenzyloxycarbonyl (“DDZ”) is used. In oneembodiment, a nitro aromatic compound containing a benzylic hydrogenortho to the nitro group is used, i.e., a chemical of the form:

where R₁ is alkoxy, alkyl, halo, aryl, alkenyl, or hydrogen; R₂ isalkoxy, alkyl, halo, aryl, nitro, or hydrogen; R₃ is alkoxy, alkyl,halo, nitro, aryl, or hydrogen; R₄ is alkoxy, alkyl, hydrogen, aryl,halo, or nitro; and R₅ is alkyl, alkynyl, cyano, alkoxy, hydrogen, halo,aryl, or alkenyl. Other materials which may be used includeo-hydroxy-α-methyl cinnamoyl derivatives. Photoremovable protectivegroups are described in, for example, Patchornik, J. Am. Chem. Soc.92:6333 (1970) and Amit et al., J. Org. Chem. 39:192 (1974), both ofwhich are hereby incorporated by reference.

In an alternative embodiment, the positive reactive group is activatedfor reaction with reagents in solution. For example, a 5-bromo-7-nitroindoline group, when bound to a carbonyl, undergoes reaction uponexposure to light at 420 nm.

In a second alternative embodiment, the reactive group on the linkermolecule is selected from a wide variety of negative light-reactivegroups including a cinnamete group.

Alternatively, the reactive group is activated or deactivated byelectron beam lithography, x-ray lithography, or any other radiation. Asuitable reactive group for electron beam lithography is a sulfonylgroup. Other methods may be used including, for example, exposure to acurrent source. Other reactive groups and methods of activation may beused in view of this disclosure.

The linking molecules are preferably exposed to, for example, lightthrough a suitable mask using photolithographic techniques of the typeknown in the semiconductor industry and described in, for example, Sze,VLSI Technology, McGraw-Hill (1983), and Mead et al., Introduction toVLSI Systems, Addison-Wesley (1980), which are hereby incorporated byreference for all purposes. The light may be directed at either thesurface containing the protective groups or at the back of thesubstrate, so long as the substrate is transparent to the wavelength oflight needed for removal of the protective groups.

The mask is in one embodiment a transparent support material selectivelycoated with a layer of opaque material. Portions of the opaque materialare removed, leaving opaque material in the precise pattern desired onthe substrate surface. The mask is brought directly into contact withthe substrate surface. “Openings” in the mask correspond to locations onthe substrate where it is desired to remove photoremovable protectivegroups from the substrate. Alignment may be performed using conventionalalignment techniques in which alignment marks are used accurately tooverlay successive masks with previous patterning steps, or moresophisticated techniques may be used. For example, interferometrictechniques such as the one described in Flanders et al., “A NewInterferometric Alignment Technique.” App. Phys. Lett. 31:426-428(1977), which is hereby incorporated by reference, may be used.

To enhance contrast of light applied to the substrate, it is desirableto provide contrast enhancement materials between the mask and thesubstrate according to some embodiments. This contrast enhancement layermay comprise a molecule which is decomposed by light such as quinonediazide or a material which is transiently bleached at the wavelength ofinterest. Transient bleaching of materials will allow greaterpenetration where light is applied, thereby enhancing contrast.Alternatively, contrast enhancement may be provided by way of a claddedfiber optic bundle.

The light may be from a conventional incandescent source, a laser, alaser diode, or the like. If non-collimated sources of light are used,it may be desirable to provide a thick- or multi-layered mask to preventspreading of the light onto the substrate. It may, further, be desirablein some embodiments to utilize groups which are sensitive to differentwavelengths to control synthesis. For example, by using groups which aresensitive to different wavelengths, it is possible to select branchpositions in the synthesis of a polymer or eliminate certain maskingsteps.

Alternatively, the substrate may be translated under a modulated laseror diode light source. Such techniques are discussed in, for example,U.S. Pat. No. 4,719,615 to Feyrer et al., which is hereby incorporatedby reference. In alternative embodiments, a laser galvanometric scanneris utilized. In other embodiments, the synthesis may take place on or incontact with a conventional liquid crystal (referred to herein as a“light valve”) or fiber optic light sources. By appropriately modulatingliquid crystals, light may be selectively controlled to permit light tocontact selected regions of the substrate. Alternatively, synthesis maytake place on the end of a series of optical fibers to which light isselectively applied. Other means of controlling the location of lightexposure will be apparent to those of skill in the art.

The development of linkers and handles for peptide synthesis isdescribed in Fields, et al., “Principles and Practice of Solid-PhasePeptide Synthesis,” Synthetic Peptides: A User's Guide, G. Grant,Editor. W.H. Freeman and Co.: New York. p. 77-183 (1992); G. Barany, etal., “Recent Progress on Handles and Supports for Solid-phase PeptideSynthesis”, Peptides-Chemistry, Structure and Biology: Proceedings ofthe Thirteenth American Peptide Symposium, R. S. Hodges and J. A. Smith,Editor. Escom Science Publishers: Leiden, The Netherlands pp. 1078-80(1994), which are hereby incorporated by reference. This technology isreadily extendable to DNA and PNA. Of particular interest is thedevelopment of PAL (Albericio, et al., “Preparation and Application ofthe5-(4-(9-Fluorenylmethyloxycarbonyl)Aminomethyl-3,5-Dimethoxyphenoxy)ValericAcid (PAL) Handle for the Solid-phase Synthesis of C-terminal PeptideAmides under Mild Conditions,” J. Org. Chem., 55:3730-3743 (1990), whichis hereby incorporated by reference, and ester (HAL) (Albericio, et al.,“Hypersensitive Acid-labile (HAL) Tris(alkoxy)Benzyl Ester Anchoring forSolid-phase Synthesis of Protected Peptide Segments,” Tetrahedron Lett.,32:1015-1018 (1991), which is hereby incorporated by reference,linkages, which upon cleavage with acid provide, respectively,C-terminal peptide amides, and protected peptide acids that can be usedas building blocks for so-called segment condensation approaches. Thestabilized carbonium ion generated in acid from cleavage of PAL or HALlinkages can be intercepted by tryptophanyl-peptides. While thisreaction is a nuisance for peptide synthesis and preventable (in part)by use of appropriate scavengers, it has the positive application ofchemically capturing oligo-Trp-end-labelled DNA and PNA molecules byHAL-modified surfaces.

The art recognizes several approaches to making oligonucleotide arrays.Southern, et al., “Analyzing and Comparing Nucleic Acid Sequences byHybridization to Arrays of Oligonucleotides: Evaluation usingExperimental Models,” Genomics, 13:1008-1017 (1992); Fodor, et al.,“Multiplexed Biochemical Assays with Biological Chips,” Nature,364:555-556 (1993); Khrapko, et al., “A Method for DNA Sequencing byHybridization with Oligonucleotide Matrix,” J. DNA Seq. Map., 1:375-388(1991); Van Ness, et al., “A Versatile Solid Support System forOligodeoxynucleoside Probe-based Hybridization Assays,” Nucleic AcidsRes., 19:3345-3350 (1991); Zhang, et al., “Single-base MutationalAnalysis of Cancer and Genetic Diseases Using Membrane Bound ModifiedOligonucleotides,” Nucleic Acids Res., 19:3929-3933 (1991); K. Beattie,“Advances in Genosensor Research,” Clin. Chem. 41(5):700-06 (1995),which are hereby incorporated by reference. These approaches may bedivided into three categories: (i) Synthesis of oligonucleotides bystandard methods and their attachment one at a time in a spatial array;(ii) Photolithographic masking and photochemical deprotection on asilicon chip, to allow for synthesis of short oligonucleotides (Fodor,et al., “Multiplexed Biochemical Assays with Biological Chips,” Nature,364:555-556 (1993) and R. J. Lipshutz, et al., “Using OligonucleotideProbe Arrays To Assess Genetic Diversity,” Biotechniques 19:442-447(1995), which are hereby incorporated by reference); and (iii) Physicalmasking to allow for synthesis of short oligonucleotides by addition ofsingle bases at the unmasked areas (Southern, et al., “Analyzing andComparing Nucleic Acid Sequences by Hybridization to Arrays ofOligonucleotides: Evaluation Using Experimental Models,” Genomics,13:1008-1017 (1992); Maskos, et al., “A Study of OligonucleotideReassociation Using Large Arrays of Oligonucleotides Synthesised on aGlass Support,” Nucleic Acids Res., 21:4663-4669 (1993), which arehereby incorporated by reference).

Although considerable progress has been made in constructingoligonucleotide arrays, some containing as many as 256 independentaddresses, these procedures are less preferred, for detecting specificDNA sequences by hybridizations. More particularly, arrays containinglonger oligonucleotides can currently be synthesized only by attachingone address at a time and, thus, are limited in potential size. Currentmethods for serially attaching an oligonucleotide take about 1 hour,thus an array of 1,000 addresses would require over 40 days ofaround-the-clock work to prepare. Arrays containing shortoligonucleotides of 8- to 10-mers do not have commercial applicability,because longer molecules are needed to detect single base differenceseffectively.

These prior procedures may still be useful to prepare said supportscarrying an array of oligonucleotides for the method of detection of thepresent invention. However, there are more preferred approaches.

It is desirable to produce a solid support with a good loading ofoligonucleotide or PNA oligomer in a relatively small, but well-definedarea. Current, commercially available fluorescent imagers can detect asignal as low as 3 attomoles per 50 μm square pixel. Thus, a reasonablesize address or “spot” on an array would be about 4×4 pixels, or 200 μmsquare. Smaller addresses could be used with CCD detection. The limit ofdetection for such an address would be about 48 attomoles per “spot”,which is comparable to the 100 attomole detection limit using afluorescent DNA sequencing machine. The capacity of oligonucleotideswhich can be loaded per 200 μm square will give an indication of thepotential signal to noise ratio. A loading of 20 fmoles would give asignal to noise ratio of about 400 to 1, while 200 fmoles would allowfor a superb signal to noise ratio of about 4000 to 1. Theoligonucleotide or PNA oligomer should be on a flexible “linker arm” andon the “outside” or “surface” of the solid support for easierhybridizations. The support should be non-fluorescent, and should notinterfere with hybridization nor give a high background signal due tononspecific binding.

The complementary capture oligonucleotide addresses on the solidsupports can be either DNA or PNA. PNA-based capture is preferred overDNA-based capture, because PNA/DNA duplexes are much stronger thanDNA/DNA duplexes, by about 1° C./base-pair. M. Egholm, et al., “PNAHybridizes to Complementary Oligonucleotides Obeying the Watson-CrickHydrogen-bonding Rules,” Nature, 365:566-568 (1993), which is herebyincorporated by reference. Thus, for a 24-mer DNA/DNA duplex withT_(m)=72° C., the corresponding duplex with one PNA strand would have a“predicted” T_(m)=96° C. (the actual melting point might be slightlylower as the above “rule of thumb” is less accurate as melting pointsget over 80° C.). Additionally, the melting difference between DNA/DNAand PNA/DNA becomes even more striking at low salt.

The melting temperature of DNA/DNA duplexes can be estimated as[4n(G·C)+2m(A·T)]° C. Oligonucleotide capture can be optimized bynarrowing the T_(m) difference between duplexes formed by captureoligonucleotides and the complementary addressable array-specificportions hybridized to one another resulting from differences in G·C/A·Tcontent. Using 5-propynyl-dU in place of thymine increases the T_(m) ofDNA duplexes an average of 1.7° C. per substitution. Froehler, et al.,“Oligonucleotides Containing C-5 Propyne Analogs of 2′-deoxyuridine and2′-deoxycytidine,” Tetrahedron Lett., 33:5307-5310 (1992) and J. Sagi,et al., Tetrahedron Letters, 34:2191 (1993), which are herebyincorporated by reference. The same substitution in the capture schemeshould lower the T_(m) difference between the components of suchduplexes and raise the T_(m) for all of the duplexes. Phosphoramiditederivatives of 5-propynyl-dU having the following structure can beprepared according to the immediately preceding Froehler and Sagireferences, which are hereby incorporated by reference.

The 5-propynyluracil PNA monomer with Fmoc amino protection can be madeby the following synthesis (where DMF is N,N′-dimethylformamide, DCC isN,N′-dicyclohexylcarbodiimide, HOBt is 1-hydroxybenzotriazole, and THFis tetrahydrofuran):

Using the methods described by Egholm, et al., “Peptide Nucleic Acids(PNA). Oligonucleotide Analogues with an Achiral Peptide Backbone,” J.Am. Chem. Soc., 114:1895-1897 (1992) and Egholm, et al., “Recognition ofGuanine and Adenine in DNA by Cytosine and Thymine Containing PeptideNucleic Acids (PNA),” J. Am. Chem. Soc., 114:9677-9678 (1992), which arehereby incorporated by reference. The synthesis scheme above describesthe preparation of a PNA monomer having a 5-propynyl-uracil basecomponent. 5-Iodouracil is first alkylated with iodoacetic acid, and,then, the propynl group is coupled to the base moiety by a Pd/Cucatalyst. The remaining steps in the scheme follow from theabove-referenced methods. These monomers can be incorporated intosynthetic DNA and PNA strands.

There are two preferred general approaches for synthesizing arrays. Inthe first approach, full-length DNA oligonucleotides or PNA oligomersare prepared and are subsequently linked covalently to a solid supportor membrane. In the second approach, specially designed PNA oligomers orDNA oligonucleotides are constructed by sequentially adding multimers tothe solid support. These multimers are added to specific rows or columnson a solid support or membrane surface. The resulting “checkerboard”pattern generates unique addressable arrays of full length PNA or DNA.

FIGS. 14-16 show different modes of preparing full-length DNAoligonucleotides or PNA oligomers and, subsequently, linking those fulllength molecules to the solid support.

FIGS. 14A-E depict a method for constructing an array of DNA or PNAoligonucleotides by coupling individual full-length oligonucleotides tothe appropriate locations in a grid. The array of FIG. 14A shows thepattern of oligonucleotides that would be generated if oligonucleotidesare coupled to sixteen 200 μm×200 μm regions of the array surface. Eachindividual 200 μm×200 μm region contains DNA or PNA with a uniquesequence which is coupled to the surface. The individual square regionswill be separated from adjacent squares by 200 μm. The array of FIG. 14Acan thus support 64 (8×8) different oligonucleotides in an 3 mm by 3 mmarea. In order to multiplex the construction of the array, 16 squaresseparated by a distance of 800 μm will be coupled simultaneously totheir specific oligonucleotides. Therefore, the 8×8 grid could beconstructed with only 4 machine steps as shown in FIGS. 14B-14E. Inthese diagrams, the dark squares represent locations that are beingaltered in the current synthesis step, while the hatched squaresrepresent regions which have been synthesized in earlier steps. Thefirst step (FIG. 14B) would immobilize oligonucleotides at locations A1,E1, I1, M1, A5, E5, I5, M5, A9, E9, I9, M9, A13, E13, I13, and M13simultaneously. In the next step (FIG. 14C), the machine would berealigned to start in Column C. After having completed Row 1, the nextstep (FIG. 14D) would start at Row 3. Finally, the last 16oligonucleotides would be immobilized in order to complete the 8×8 grid(FIG. 14E). Thus, the construction of the 8×8 array could be reduced to4 synthesis steps instead of 64 individual spotting reactions. Thismethod would be easily extended and an apparatus capable of spotting 96oligomers simultaneously could be used rapidly to construct largerarrays.

FIGS. 15A-E represent a perspective dimensional view of the arrayconstruction process described in FIG. 14. In FIGS. 15A-E, theconstruction of a 4×4 (16) array using a machine capable of spottingfour different 24-mers simultaneously is depicted. First, as shown inFIG. 15A, the machine attaches 4 oligomers at locations A1, E1, A5, andE5. Next, as shown in FIG. 15B, the machine is shifted horizontally andattaches 4 oligomers at locations C1, G1, C5, and G5. Next, as shown inFIG. 15C, the machine is repositioned and attaches 4 oligomers atlocations A3, E3, A7, and E7. Finally, as shown in FIG. 15D, the machineattaches the 4 remaining oligomers at positions C3, G3, C7, and G7. Thecompleted array contains sixteen 24-mers as shown in the perspectiveview of FIG. 15E.

FIGS. 16A-C show views for an application apparatus 2 capable ofsimultaneously coupling 16 different oligonucleotides to differentpositions on an array grid G as shown in FIG. 14. The black squares inthe top view (FIG. 16A) represent sixteen 200 μm×200 μm regions that arespatially separated from each other by 600 μm. The apparatus shown has16 input ports which would allow tubes containing differentoligonucleotides to fill the funnel-shaped chambers above differentlocations on the array. The side views (FIGS. 16B-C, taken along lines16B-16B and 16C-16C, of FIG. 16A, respectively) of the apparatusdemonstrate that funnel shaped chambers 4 align with the appropriateregion on the array below the apparatus. In addition, two valves 6 and 8(hatched-squares in FIGS. 16A-C) control the flow of fluid in the 16independent reaction chambers. One valve 6 controls the entry of fluidsfrom the input port 10, while the other valve 8 would be attached to avacuum line 12 in order to allow loading and clearing of the reactionchamber 4. The apparatus would first be aligned over the appropriate 200μm×200 μm regions of the array. Next, the entire apparatus is firmlypressed against the array, thus forming a closed reaction chamber aboveeach location. The presence of raised 10 μm ridges R around eachlocation on the array ensures the formation of a tight seal which wouldprevent leakage of oligomers to adjacent regions on the array. Next, thevalve 8 to vacuum line 12 would be opened, while the valve 10 from theinput solution port 10 would be closed. This would remove air from thereaction chamber 4 and would create a negative pressure in the chamber.Then, the valve 8 to vacuum line 12 would be closed and the valve 10from the input solution port 10 would be opened. The solution would flowinto the reaction chamber 4 due to negative pressure. This processeliminates the possibility of an air bubble forming within the reactionchamber 4 and ensures even distribution of oligonucleotides across the200 μm×200 μm region. After the oligonucleotides have been coupled tothe activated array surface, the input valve 6 would be closed, thevalve 8 to vacuum line 12 would be opened, and the apparatus would belifted from the array surface in order to remove completely any excesssolution from the reaction chamber. A second apparatus can now berealigned for the synthesis of the next 16 locations on the array.

FIGS. 15 to 26 show different modes of constructing PNA oligomers or DNAoligonucleotides on a solid support by sequentially adding,respectively, PNA or DNA, multimers to the solid support.

As an example of assembling arrays with multimers, such assembly can beachieved with tetramers. Of the 256 (4⁴) possible ways in which fourbases can be arranged as tetramers, 36 that have unique sequences can beselected. Each of the chosen tetramers differs from all the others by atleast two bases, and no two dimers are complementary to each other.Furthermore, tetramers that would result in self-pairing or hairpinformation of the addresses have been eliminated.

The final tetramers are listed in Table 1 and have been-numberedarbitrarily from 1 to 36. This unique set of tetramers are used asdesign modules for the sometimes desired 24-mer capture oligonucleotideaddress sequences. The structures can be assembled by stepwise (one baseat a time) or convergent (tetramer building blocks) syntheticstrategies. Many other sets of tetramers may be designed which followthe above rules. The segment approach is not uniquely limited totetramers, and other units, i.e. dimers, trimers, pentamers, or hexamerscould also be used.

TABLE 1 List of tetramer PNA sequences and complementary DNA sequences,which differ from each other by at least 2 bases. Number Sequence (N-C)Complement (5′-3′) G + C 1. TCTG CAGA 2 2. TGTC GACA 2 3. TCCC GGGA 3 4.TGCG CGCA 3 5. TCGT ACGA 2 6. TTGA TCAA 1 7. TGAT ATCA 1 8. TTAG CTAA 19. CTTG CAAG 2 10. CGTT AACG 2 11. CTCA TGAG 2 12. CACG CGTG 3 13. CTGTACAG 2 14. CAGC GCTG 3 15. CCAT ATGG 2 16. CGAA TTCG 2 17. GCTT AAGC 218. GGTA TACC 2 19. GTCT AGAC 2 20. GACC GGTC 3 21. GAGT ACTC 2 22. GTGCGCAC 3 23. GCAA TTGC 2 24. GGAC GTCC 3 25. AGTG CACT 2 26. AATC GATT 127. ACCT AGGT 2 28. ATCG CGAT 2 29. ACGG CCGT 3 30. AGGA TCCT 2 31. ATACGTAT 1 32. AAAG CTTT 1 33. CCTA TAGG 2 34. GATG CATC 2 35. AGCC GGCT 336. TACA TGTA 1Note that the numbering scheme for tetramers permits abbreviation ofeach address as a string of six numbers (e.g., second column of Table 2infra). The concept of a 24-mer address designed from a unique set of 36tetramers (Table 1) allows a huge number of possible structures,36⁶=2,176,782,336.

FIG. 17 shows one of the many possible designs of 36 tetramers whichdiffer from each other by at least 2 bases. The checkerboard patternshows all 256 possible tetramers. A given square represents the firsttwo bases on the left followed by the two bases on the top of thecheckerboard. Each tetramer must differ from each other by at least twobases, and should be non-complementary. The tetramers are shown in thewhite boxes, while their complements are listed as (number). Thus, thecomplementary sequences GACC (20) and GGTC (20′) are mutually exclusivein this scheme. In addition, tetramers must be non-palindromic, e.g.,TCGA (darker diagonal line boxes), and non-repetitive, e.g., CACA(darker diagonal line boxes from upper left to lower right). All othersequences which differ from the 36 tetramers by only 1 base are shadedin light gray. Four potential tetramers (white box) were not chosen asthey are either all A·T or G·C bases. However, as shown below, the T_(m)values of A·T bases can be raised to almost the level of G·C bases.Thus, all A·T or G·C base tetramers (including the ones in white boxes)could potentially be used in a tetramer design. In addition, thymine canbe replaced by 5-propynyl uridine when used within captureoligonucleotide address sequences as well as in the oligonucleotideprobe addressable array-specific portions. This would increase the T_(m)of an A·T base pair by ˜1.7° C. Thus, T_(m) values of individualtetramers should be approximately 15.1° C. to 15.7° C. T_(m) values forthe full length 24-mers should be 95° C. or higher.

To illustrate the concept, a subset of six of the 36 tetramer sequenceswere used to construct arrays: 1=TGCG; 2=ATCG; 3=CAGC; 4=GGTA; 5=GACC;and 6=ACCT. This unique set of tetramers can be used as design modulesfor the required 24-mer addressable array-specific portion and 24-mercomplementary capture oligonucleotide address sequences. This embodimentinvolves synthesis of five addressable array-specific portion (sequenceslisted in Table 2). Note that the numbering scheme for tetramers allowsabbreviation of each portion (referred to as “Zip #”) as a string of sixnumbers (referred to as “zip code”).

TABLE 2 List of all 5 DNA/PNA oligonucleotide address sequences. Zip #Zip code Sequence (5′ → 3′ or NH₂ → COOH) G + C Zip 11 1-4-3-6-6-1TGCG-GGTA-CAGC-ACCT-ACCT-TGCG 15 (SEQ ID NO: 2) Zip 12 2-4-4-6-1-1ATCG-GGTA-GGTA-ACCT-TGCG-TGCG 14 (SEQ ID NO: 3) Zip 13 3-4-5-6-2-1CAGC-GGTA-GACC-ACCT-ATCG-TGCG 15 (SEQ ID NO: 4) Zip 14 4-4-6-6-3-1GGTA-GGTA-ACCT-ACCT-CAGC-TGCG 14 (SEQ ID NO: 5) Zip 15 5-4-1-6-4-1GACC-GGTA-TGCG-ACCT-GGTA-TGCG 15 (SEQ ID NO: 6)Each of these oligomers contains a hexaethylene oxide linker arm ontheir 5′ termini [P. Grossman, et al., Nucl. Acids Res., 22:4527-4534(1994), which is hereby incorporated by reference], and ultimateamino-functions suitable for attachment onto the surfaces of glassslides, or alternative materials. Conjugation methods will depend on thefree surface functional groups [Y. Zhang, et al., Nucleic Acids Res.,19:3929-3933 (1991) and Z. Guo, et al., Nucleic Acids Res., 34:5456-5465(1994), which are hereby incorporated by reference].

Synthetic oligonucleotides (normal and complementary directions, eitherfor capture hybridization or hybridization/ligation) are prepared aseither DNA or PNA, with either natural bases or nucleotide analogues.Such analogues pair with perfect complementarity to the natural basesbut increase T_(m) values (e.g., 5-propynyl-uracil).

Each of the capture oligonucleotides have substantial sequencedifferences to minimize any chances of cross-reactivity—see FIG. 17 andTable 1. Rather than carrying out stepwise synthesis to introduce basesone at a time, protected PNA tetramers can be used as building blocks.These are easy to prepare; the corresponding protected oligonucleotideintermediates require additional protection of the internucleotidephosphate linkages. Construction of the 24-mer at any given addressrequires only six synthetic steps with a likely improvement in overallyield by comparison to stepwise synthesis. This approach eliminatestotally the presence of failure sequences on the support, which couldoccur when monomers are added one-at-a-time to the surface. Hence, incontrast to previous technologies, the possibilities for false signalsare reduced. Moreover, since failure sequences at each address areshorter and lacking at least four bases, there is no risk that thesewill interfere with correct hybridization or lead to incorrecthybridizations. This insight also means that “capping” steps will not benecessary.

Masking technology will allow several addresses to be built upsimultaneously, as is explained below. As direct consequences of themanufacturing process for the arrays, several further advantages arenoted. Each 24-mer address differs from its nearest 24-mer neighbor bythree tetramers, or at least 6 bases. At low salt, each base mismatch inPNA/DNA hybrids decreases the melting temperature by 8° C. Thus, theT_(m) for the correct PNA/DNA hybridization is at least 48° C. higherthan any incorrect hybridization. Also, neighboring 24-mers areseparated by 12-mers, which do not hybridize with anything and represent“dead” zones in the detection profile. PNA addresses yield rugged,reusable arrays.

The following description discloses the preparation of 36 unique PNAtetramers and shows the mechanical/chemical strategy to prepare thearrays. This technique can be used to create a 5×5 array with 25addresses of PNA 24-mers. Alternatively, all 36 tetramers can beincorporated to generate full-size arrays of 1,296 addresses.

FIGS. 18A-G are schematic diagrams showing addition of PNA tetramers togenerate a 5×5 array of unique 24 mer addresses. The manufacturingdevice is able to add PNA tetramers in either columns, or in rows, byrotating the multi-chamber device or surface 90°. A circular manifoldallows circular permutation of tetramer addition. Thus, complex uniqueaddresses may be built by using a simple algorithm. In the firsttetramer addition, PNA tetramers 1, 2, 3, 4, and 5 are linked to thesurface in each of the 5 columns, respectively as shown in FIG. 18A.After rotating the chamber 90°, PNA tetramers 6, 5, 4, 3, and 2 areadded in adjacent rows, as shown in FIG. 18B. In the third step, asshown in FIG. 18C, tetramers 3, 4, 5, 6, and 1 (note circularpermutation) are added in columns. In the 4th step, as shown in FIG.18D, tetramers 2, 1, 6, 5, and 4 are added in adjacent rows, etc. Thisprocess continues in the manner shown in FIGS. 23E-G described infra.The bottom of the diagram depicts tetramer sequences which generateunique 24 mers at each position. The middle row of sequences1-4-3-6-6-1; 2-4-4-6-1-1; 3-4-5-6-2-1; 4-4-6-6-3 and 5-4-1-6-4-1 areshown in full length in Table 2. The addition of tetramers in acircularly permuted fashion can be used to generate larger arrays.Tetramer addition need not be limited to circular patterns and can beadded in many other combinations to form unique addresses which differfrom each other by at least 3 tetramers, which translates to at least 6bases.

The present invention has greater specificity than existing mutationdetection methods which use allele-specific PCR, differentialhybridization, or sequencing-by-hybridization. These methods rely onhybridization alone to distinguish single-base differences in twootherwise identical oligonucleotides. The signal-to-noise ratios forsuch hybridization are markedly lower than those that can be achievedeven with the two most closely-related capture oligonucleotides in anarray. Since each address is designed by alternating tetramer additionin three rows and three columns, a given address will differ by at leastthree tetramers from its neighbor. Since each tetramer differs fromevery other tetramer by at least 2 bases, a given address will differfrom another address by at least 6 bases. However, in practice, mostaddresses will differ from most other addresses by considerably morebases.

This concept is illustrated below using the two addresses, Zip 12 andZip 14. These two addresses are the most related among the 25 addressesschematically represented in FIGS. 18 and 20 (discussed infra). Thesetwo addresses have in common tetramers on every alternating position(shown as underlined):

Zip 12 (2-4-4-6-1-1) = 24 mer 5′-ATCG GGTA GGTA ACCT TGCG TGCG-3′(SEQ IDNO: 7) Zip 14 (4-4-6-6-3-1) = 24 mer 5′-GGTA GGTA ACCT ACCT CAGCTGCG-3′(SEQ ID NO: 8)

In addition, they have in common a string of 12 nucleotides, as well asthe last four in common (shown as underlined):

Zip 12 (2-4-4-6-1-1) = 24 mer 5′-ATCG GGTA GGTA ACCT TGCG TGCG-3′(SEQ IDNO: 9) Zip 14 (4-4-6-6-3-1) = 24 mer 5′-GGTA GGTA ACCT ACCT CAGCTGCG-3′(SEQ ID NO: 10)

Either representation has at least 8 differences between theoligonucleotides. Although an oligonucleotide complementary to Zip 12 orZip 14 at the underlined nucleotides could hybridize to both of theseaddresses at a lower temperature (e.g., 37° C.), only the fullycomplementary oligonucleotide would hybridize at elevated temperature(e.g., 70° C.).

Furthermore, for other capture oligonucleotides, such as Zip 3, thenumber of shared nucleotides is much lower (shown as underlined):

Zip 12 (2-4-4-6-1-1) = 24 mer 5′-ATCG GGTA GGTA ACCT TGCG TGCG-3′(SEQ IDNO: 11) Zip 3 (3-6-5-2-2-3) = 24 mer 5′-CAGC ACCT GACC ATCG ATCGCAGC-3′(SEQ ID NO: 12)

Therefore, the ability to discriminate Zip 12 from Zip 3 duringhybridization is significantly greater than can be achieved using any ofthe existing methods.

A multi-chamber device with alternating chambers and walls (each 200 μmthick) will be pressed onto the modified glass or silicon surface ofFIG. 19A prior to delivery of PNA tetramers into either columns or rows.The surface will be etched to produce 10 μm ridges (black lines) toeliminate leaking between chambers. Initially, a flexible spacer(linker) is attached to the array surface. In the first step, as shownin FIG. 19B, PNA tetramers, 1, 2, 3, 4, and 5 are linked to the surfacein each of the five columns, respectively. The multi-chamber device isthen rotated 90°. Tetramers 6, 5, 4, 3, and 2 are added in adjacentrows, as shown in FIG. 19C. The process is repeated a total of threetimes to synthesize 24-mer PNA oligomers. Each completed 24-mer within agiven row and column represents a unique PNA sequence, hence achievingthe desired addressable array. Smaller oligonucleotide sequencesrepresent half-size 12-mers which result from 3 rounds of synthesis inthe same direction. Since each 24-mer differs from its neighbor by threetetramers and each tetramer differs from another by at least 2 bases,then each 24-mer differs from the next by at least 6 bases (i.e., 25% ofthe nucleotides differ). Thus, a wrong address would have 6 mismatchesin just 24 bases and, therefore, would not be captured at the wrongaddress, especially under 75-80° C. hybridization conditions. Inaddition, while a particular smaller 12-mer sequence may be found withina 24-mer sequence elsewhere on the grid, an addressable array-specificportion will not hybridize to the 12-mer sequence at temperatures above50° C.

The starting surfaces will contain free amino groups, a non-cleavableamide linkage will connect the C-terminus of PNA to the support, andorthogonal side-chain deprotection must be carried out upon completionof segment condensation assembly in a way that PNA chains are retainedat their addresses. A simple masking device has been designed thatcontains 200 μm spaces and 200 μm barriers, to allow each of 5 tetramersto couple to the solid support in distinct rows (FIG. 20A). Afteraddition of the first set of tetramers, the masking device is rotated90°, and a second set of 5 tetramers are added (FIG. 20B). This can becompared to putting icing on a cake as rows, followed by icing ascolumns. The intersections between the rows and columns will containmore icing, likewise, each intersection will contain an octamer ofunique sequence. Repeating this procedure for a total of 6 cyclesgenerates 25 squares containing unique 24-mers, and the remainingsquares containing common 12-mers (FIGS. 20C and 21A-G). The silicon orglass surface will contain 10 μm ridges to assure a tight seal, andchambers will be filled under vacuum. A circular manifold (FIG. 26) willallow for circular permutation of the six tetramers prior to deliveryinto the five rows (or columns). This design generates unique 24-merswhich always differ from each other by at least 3 tetramers, even thoughsome sequences contain the same 3 tetramers in a contiguous sequence.This masking device is conceptually similar to the masking techniquedisclosed in Southern, et al., Genomics, 13:1008-1017 (1992) and Maskos,et al., Nucleic Acids Res., 21:2267-2268 (1993), which are herebyincorporated by reference, with the exception that the array is builtwith tetramers as opposed to monomers.

Alternatively, the production of the incomplete 12-mer sequences can beeliminated if a mask which isolates each location is used. In the firststep (as shown in FIG. 19D), PNA tetramers 1, 2, 3, 4, and 5 are linkedto the surface in each of the five columns respectively. Themulti-chamber device is then rotated 90°. Tetramers 6, 5, 4, 3, and 2are added in adjacent rows, as shown in FIG. 19E. The process isrepeated a total of three times to synthesize 24-mer PNA oligomers. Eachcompleted 24-mer within a given row and column represents a unique PNAsequence, hence achieving the desired addressable array. In addition,each 24-mer will be separated from adjacent oligomers by a 200 μm regionfree of PNA oligomers.

A silicon or glass surface will be photochemically etched to produce acrosshatched grid of 10 μm raised ridges in a checkerboard pattern (seeFIG. 20). Alternate squares (200 μm×200 μm) will be activated, allowingfor the attachment of C₁₈ alkyl spacers and PEG hydrophilic linkers (MW400-4,000), such that each square is separated by at least one blanksquare and two ridges on all sides.

An example of a universal array using PNA tetramers can be formed byadding 36 different tetramers to either 36 columns or rows at the sametime. The simplest way to add any tetramer to any row is to have all 36tetramer solutions attached by tubings to minivalves to a circularmanifold which has only one opening. The other side of the circularmanifold can be attached to any of 36 minivalves which go to individualrows (or columns). So by rotating the manifold and minivalves to thechambers (rows), one can pump any tetramer into any row, one at a time.This can be either a mechanical device requiring physical rotation or,alternatively, can be accomplished by using electronic microvalves alonga series of import (tetramers) and export (rows) channels. This processcan occur quite rapidly (5 seconds, including rinsing out the manifoldfor its next use), so that it would take about 36×5=180 sec. to add all36 rows.

A potentially more rapid way of filling the rows or columns, would be tofill all of them simultaneously. This is illustrated in FIG. 20 for a5×5 array. The silicon or glass surface will contain 10 μm ridges toassure a tight seal, and chambers will be filled using the vacuumtechnique described above. A circular manifold will allow for circularpermutation of the six tetramers prior to delivery into the five rows(or columns). In FIG. 20, the first step is 5, 4, 3, 2, 1. When rotatingthe multi-chamber device, one could continue to add in either numerical,or reverse numerical order. In the example, a numerical order of 2, 3,4, 5, 6 for the second step is used. In the third step, the circularpermutation (reverse) gives 1, 6, 5, 4, 3. Fourth step (forward) 4, 5,6, 1, 2. Fifth step (reverse) 4, 3, 2, 1, 6. Sixth step (forward) 5, 6,1, 2, 3. This can be expanded to 36 tetramers into 36 rows (or columns).This approach limits the potential variations in making the address forthe array from 36⁶=2,176,782,336 in every position to 36⁶=2,176,782,336in one position, with the other 1,295 positions now defined by the firstaddress. This is still a vast excess of the number of differentaddresses needed. Furthermore, each address will still differ from everyother address by at least 6 nucleotides.

Note that all of these arrays can be manufactured in groups, just asseveral silicon chips can be produced on the same wafer. This isespecially true of the tetramer concept, because this requires addingthe same tetramer in a given row or column. Thus, one row could cover aline of ten arrays, so that a 10×10 grid=100 arrays could bemanufactured at one time.

Alternatively, the process described with reference to FIG. 20 can becarried out with one less cycle to make a 20 mer oligonucleotide. Thecapture oligonucleotide should be sufficiently long to capture itscomplementary addressable array-specific portion under the selectedhybridization conditions.

FIGS. 21A-F show a schematic cross-sectional view of the synthesis of anaddressable array (legend). FIG. 21A shows attachment of a flexiblespacer (linker) to surface of array. FIG. 21B shows the synthesis of thefirst rows of oligonucleotide tetramers. Only the first row, containingtetramer 1, is visible. A multi-chamber device is placed so thatadditional rows, each containing a different tetramer, are behind thefirst row. FIG. 21C shows the synthesis of the first columns ofoligonucleotide tetramers. The multi-chamber device or surface has beenrotated 90°. Tetramers 9, 18, 7, and 12 were added in adjacent chambers.FIG. 21D shows the second round synthesis of the oligonucleotide rows.The first row contains tetramer 2. FIG. 21E shows the second round ofsynthesis of oligonucleotides. Tetramers 34, 11, 14, and 23 are added inadjacent chambers during the second round. FIG. 21F shows the thirdround synthesis of PNA rows. The first row contains tetramer 3. FIG. 21Fshows the structure of the array after third round synthesis of columns,adding tetramers 16, 7, 20, 29. Note that all 24-mer oligonucleotideswithin a given row or column are unique, hence achieving the desiredaddressable array. Since each 24-mer differs from its neighbor by threetetramers, and tetramers differ from each other by at least 2 bases,then each 24-mer differs from the next by at least 6 bases. Eachmismatch significantly lowers T_(m), and the presence of 6 mismatches injust 24 bases would make cross hybridization unlikely even at 35° C.Note that the smaller 12-mer sequences are identical with one another,but are not at all common with the 24-mer sequences. Even though theparticular 12-mer sequence may be found within a 24-mer elsewhere on thegrid, for example 17-1-2-3-28-5, an oligonucleotide will not hybridizeto the 12-mer at temperatures above 50° C.

FIGS. 22A-C present a design for a masking device 2 capable ofconstructing an array grid G as described in FIGS. 19-21. FIG. 22A is atop view of the arrangement of device 2 and array grid G, while sideviews FIGS. 22B-22C are, respectively, taken along line 22B-22B and line22C-22C of FIG. 22A. The masking device contains 200 μm spaces and 200μm barriers, to allow each of five tetramers to be coupled to the solidsupport in distinct rows. After addition of the first set of tetramers,the masking device is rotated 90°, and a second set of 5 tetramers areadded. This can be compared to putting icing on a cake as rows, followedby icing as columns. The intersections between the rows and columns willcontain more icing, likewise, each intersection will contain an octamerof unique sequence. Repeating this procedure for a total of 6 cyclesgenerates 25 spatially separated squares containing unique 24-mers, andthe remaining squares containing common 12-mers. The silicon or glasssurface will contain 10 μm ridges R to assure a tight seal, and chambers4 will be filled by opening valves 8 to a vacuum line 12 to createnegative pressure in the chamber. The multi-chamber device is pressedonto the membrane or activated solid surface, forming tight seals. Thebarriers may be coated with rubber or another material to avoid crosscontamination from one chamber to the next. One must also make sure themembrane or solid support surface is properly wetted by the solvents.After closing valves 8 to vacuum line 12, one proceeds by activating thesurface, deprotecting, and adding a tetramer to a chamber 4 throughlines 10 by opening valves 6. The chamber is unclamped, the membrane isrotated 90°, and reclamped. A second round of tetramers are added by theabove-described vacuum and tetramer application steps. A valve blockassembly (FIG. 25A-C) will route each tetramer to the appropriate row.Alternatively, a cylindrical manifold (FIGS. 26A-D) will allow circularpermutation of the six tetramers prior to delivery into the five rows(or columns). This design generates unique 24-mers which always differfrom each other by at least 3 tetramers, even though some sequencescontain the same 3 tetramers in a contiguous sequence.

FIGS. 23A-C represent a perspective view of the array constructionprocess described in FIG. 19 (FIGS. 19D-19E). In the first step, asshown in FIG. 23A, PNA tetramers 1, 2, 3, 4, and 5 are linked to thesurface in each of the five columns, respectively. Each of the 5locations in the columns are isolated, and there is a 200 μm gap betweenthem where no oligonucleotides are coupled. The multi-chamber device isthen rotated 90°, as shown in FIG. 23B. Tetramers 6, 5, 4, 3, and 2 areadded in adjacent rows. Each of the 5 locations in the rows areisolated, and there is a 200 μm gap between them where nooligonucleotides are coupled. Each completed 24-mer, as shown in FIG.23C, within a given row and column represents a unique PNA sequence.Unlike the design presented in FIGS. 19-22, this array design will notcontain the half-size 12-mers between each complete 24-mer, because amask with isolated locations will be used.

FIGS. 24A-C present a design for a masking device 2 capable ofconstructing an array grid G as described in FIG. 23. FIG. 24A is a topview of the arrangement of device 2 and array grid G, while side viewsFIGS. 24B and 24C are, respectively, taken along line 24B-24B and line24C-24C of FIG. 24A. The masking device contains 200 μm spaces and 200μm barriers, to allow each of five tetramers to be coupled to the solidsupport in distinct locations on the array grid G. After addition of thefirst set of tetramers, the masking device or surface is rotated 90°,and a second set of 5 tetramers are added. Repeating this procedure fora total of 6 cycles generates 25 spatially separated squares containingunique 24-mers, and the remaining squares containing common 12-mers. Thesilicon or glass surface will contain 10 μm ridges R to assure a tightseal, and chambers 4 will be filled by a procedure initiated by using avacuum to create negative pressure in the chamber 2. This vacuum iscreated by opening valves 8 to vacuum line 12. The multi-chamber deviceis pressed onto the membrane or activated solid surface, forming tightseals. The barriers may be coated with rubber or another material toavoid cross contamination from one chamber to the next. One must alsomake sure the membrane or solid support surface is properly wetted bythe solvents. After closing valves 8 to vacuum line 12, one proceeds byactivating the surface, deprotecting, and adding a tetramer to chamber 4through lines 10 by opening valves 6. The chamber is unclamped, themembrane is rotated 90°, and reclamped. A second round of tetramers areadded by the above-described vacuum and tetramer application steps. Avalve block assembly (FIG. 25A-C) will route each tetramer to theappropriate row. Alternatively, a cylindrical manifold (FIG. 26A-D) willallow circular permutation of the six tetramers prior to delivery intothe five rows (or columns). This design generates unique 24-mers whichare separated from each other by a region free of any oligonucleotides.

FIGS. 25A-C show a valve block assembly 14 which connects six inputports 10 to five output ports 16 via a common chamber 18. FIG. 25A is atop view of valve block assembly 14, while side views FIGS. 25B and 25Care, respectively, taken along line 25B-25B and line 25C-25C of FIG.25A. Each of the 6 input ports 10 and 5 output ports 16 contains a valve6 and a valve 20, respectively, which control the flow of fluids. The 6input tubes 10 contain different solutions, and the valve block assembly14 is capable of routing any one of the input fluids to one of the 5output ports 16 at a time. This is accomplished by opening the valve 6of one of the input ports 10 and one of the valves 20 of the outputports 16 simultaneously and allowing fluid to fill the chamber 18 andexit via the output port 16 connected to the open valve 20. The valveblock assembly 14 is connected to a source of solvent 22 and a source ofvacuum 12 via valves 24 and 8, respectively, in order to allow cleaningof the central chamber 18 in between fluid transfers. The solvent fillsthe chamber 18, and the vacuum is used to remove all fluid from thechamber. This prepares the chamber 18 for the next fluid transfer stepand prevents cross-contamination of the fluids.

FIGS. 26A-D depict a cylindrical manifold assembly 114 which transfers 6different tubes of input fluids to 5 different output tubes. Themanifold itself contains two separate halves 114A and 114B which arejoined by a common, central spoke 134 around which both halves canindependently rotate. The bottom portion 114B is a cylindrical blockwith 6 channels 130 drilled through it (see FIG. 26C, which is a bottomview of FIG. 26B taken along line 26C-26C of FIG. 26B). Each of the 6channels 130 are attached to 6 different input tubes 110. The inputtubes 110 contain valves 106 which connect the input channels 130 toeither reagents, solvent, or a vacuum via lines 138 having valves 136leading to vacuum line 112 having valve 108 and solvent line 122 havingvalve 124. This allows different fluids to enter the channels 130 and132 of the manifold and allows clearing of the channels 130 and 132 ofexcess fluid between fluid transfers. The upper portion of the manifold(see FIG. 26A, which is a top view of FIG. 26B taken along line 26A-26Aof FIG. 26B) is also a cylindrical block with 5 channels 132 drilledthrough it. The 5 channels 132 are each connected to a different outputtube 116. The two halves of manifold 114A and 114B can be independentlyrotated so that different input channels 130 will line up with differentoutput channels 132. This allows the 6 tubes of input fluids to betransferred to the 5 output tubes simultaneously. The bottom half of themanifold 114B can be rotated 60 degrees in order to align each inputport 110 with the next output port 116. In this way, each input port 110can be aligned with any of the output ports 116. The circular manifoldof FIGS. 26A-D differs from the valve block assembly of FIGS. 26A-C inthat the former can simultaneously transfer five of the six input fluidsto the five output ports, because it has 5 channels connecting inputports to the output ports. This concept could be easily expanded todeliver 36 tetramers simultaneously to 36 locations.

The present invention contains a number of advantages over prior artsystems.

The solid support containing DNA arrays, in accordance with the presentinvention, detects sequences by hybridization of ligated productsequences to specific locations on the array so that the position of thesignal emanating from captured labels identifies the presence of thesequence. For high throughput detection of specific multiplexed LDRproducts, addressable array-specific portions guide each LDR product toa designated address on the solid support. While other DNA chipapproaches try to distinguish closely related sequences by subtledifferences in melting temperatures during solution-to-surfacehybridization, the present invention achieves the required specificityprior to hybridization in solution-based LDR reactions. Thus, thepresent invention allows for the design of arrays of captureoligonucleotides with sequences which are very different from eachother. Each LDR product will have a unique addressable array-specificportion, which is captured selectively by a capture oligonucleotide at aspecific address on the solid support. When the complementary captureoligonucleotides on the solid support are either modified DNA or PNA,LDR products can be captured at higher temperatures. This provides theadded advantages of shorter hybridization times and reduced non-specificbinding. As a result, there is improved signal-to-noise ratios.

Another advantage of the present invention is that PCR/LDR allowsdetection of closely-clustered mutations, single-base changes, and shortrepeats and deletions. These are not amenable to detection byallele-specific PCR or hybridization.

In accordance with the present invention, false hybridization signalsfrom DNA synthesis errors are avoided. Addresses can be designed sothere are very large differences in hybridization T_(m) values toincorrect address. In contrast, the direct hybridization approachesdepend on subtle-differences. The present invention also eliminatesproblems of false data interpretation with gel electrophoresis orcapillary electrophoresis resulting from either DNA synthesis errors,band broadening, or false band migration.

The use of a capture oligonucleotide to detect the presence of ligationproducts, eliminates the need to detect single-base differences inoligonucleotides using differential hybridization. Other existingmethods in the prior art relying on allele-specific PCR, differentialhybridization, or sequencing-by-hybridization methods must havehybridization conditions optimized individually for each new sequencebeing analyzed. When attempting to detect multiple mutationssimultaneously, it becomes difficult or impossible to optimizehybridization conditions. In contrast, the present invention is ageneral method for high specificity detection of correct signal,independent of the target sequence, and under uniform hybridizationconditions. The present invention yields a flexible method fordiscriminating between different oligonucleotide sequences withsignificantly greater fidelity than by any methods currently availablewithin the prior art.

The array of the present invention will be universal, making it usefulfor detection of cancer mutations, inherited (germline) mutations, andinfectious diseases. Further benefit is obtained from being able toreuse the array, lowering the cost per sample.

The present invention also affords great flexibility in the synthesis ofoligonucleotides and their attachment to solid supports.Oligonucleotides can be synthesized off of the solid support and thenattached to unique surfaces on the support. Segments of multimers ofoligonucleotides, which do not require intermediate backbone protection(e.g., PNA), can be synthesized and linked onto to the solid support.Added benefit is achieved by being able to integrate these syntheticapproaches with design of the capture oligonucleotide addresses. Suchproduction of solid supports is amenable to automated manufacture,obviating the need for human intervention and resulting contaminationconcerns.

An important advantage of the array of the present invention is theability to reuse it with the previously attached captureoligonucleotides. In order to prepare the solid support for such reuse,the captured oligonucleotides must be removed without removing thelinking components connecting the captured oligonucleotides to the solidsupport. A variety of procedures can be used to achieve this objective.For example, the solid support can be treated in distilled water at95-100° C., subjected to 0.01 N NaOH at room temperature, contacted with50% dimethylformamide at 90-95° C., or treated with 50% formamide at90-95° C. Generally, this procedure can be used to remove capturedoligonucleotides in about 5 minutes. These conditions are suitable fordisrupting DNA-DNA hybridizations; DNA-PNA hybridizations require otherdisrupting conditions.

The present invention is illustrated, but not limited, by the followingexamples.

EXAMPLES Example 1 Immobilization of Capture Oligonucleotides to SolidSupports

The solid support for immobilization was glass, in particular microscopeslides. The immobilization to glass (e.g., microscope slides), or othersupports such as silicon (e.g., chips), membranes (e.g., nylonmembranes), beads (e.g., paramagnetic or agarose beads), or plasticssupports (e.g., polyethylene sheets) of capture oligonucleotides inspatially addressable arrays is comprised of 5 steps:

A. Silanization of Support

The silanization reagent was 3-aminopropyl triethoxysilane (“APTS”).Alternatively, 3-glycidoxypropyltrimethoxysilane (K. L. Beattie, et al.,“Advances in Genosensor Research,” Clin. Chem., 41:700-706 (1995); U.Maskos, et al., “Oligonucleotide Hybridizations on Glass Supports: aNovel Linker for Oligonucleotide Synthesis and Hybridization Propertiesof Oligonucleotides Synthesized in situ,” Nucleic Acids Res.,20:1679-1684 (1992); C. F. Mandenius, et al., “Coupling of Biomoleculesto Silicon Surfaces for Use in Ellipsometry and Other RelatedTechniques,” Methods Enzymol., pp. 388-394 (1988), which are herebyincorporated by reference) or 3-(trimethoxysilyl)propyl methacrylate (M.Glad, et al., “Use of Silane Monomers for Molecular Imprinting andEnzyme Entrapment in Polysiloxane-coated Porous Silica,” J. Chromatogr.347:11-23 (1985); E. Hedborg, et al., “Some Studies ofMolecularly-imprinted Polymer Membranes in Combination with Field-effectDevices,” Sensors and Actuators A 37-38:796-799 (1993); and M. Kempe, etal., “An Approach Towards Surface Imprinting Using the EnzymeRibonuclease A,” J. Mol. Recogn. 8:35-39 (1995), which are herebyincorporated by reference) can be used as an initial silanizationreagent. Prior to silanization, the support was cleansed and the surfaceof the support was rendered hydrophobic. Glass slides (FisherScientific, Extra thick microslides, frosted cat.# 12-550-11) wereincubated in conc.aq. NH₄OH—H₂O₂—H₂O (1:1:5, v/v/v) at 80° C. for 5 minand rinsed in distilled water. The support was then washed withdistilled water, ethanol and acetone as described in the literature (C.F. Mandenius, et al., “Coupling of Biomolecules to Silicon Surfaces forUse in Ellipsometry and Other Related Techniques,” Methods Enzymol., pp.388-394 (1988); Graham, et al., “Gene Probe Assays on a Fibre-OpticEvanescent Wave Biosensor,” Biosensors & Bioelectronics, 7: 487-493(1992); Jönsson, et al., “Adsorption Behavior of Fibronectin on WellCharacterized Silica Surfaces,” J. Colloid Interface Sci., 90:148-163(1982), which are hereby incorporated by reference). The support wassilanized overnight at room temperature in a solution of 2% (v/v)3-aminopropyl triethoxysilane (Sigma, St. Louis, Mo.) in dry acetone(99.7%) (modified after Z. Guo, et. al., “Direct Fluorescence Analysisof Genetic Polymorphisms by Hybridization with Oligonucleotide Arrays onGlass Supports,” Nucl. Acids Res. 22:5456-65 (1994), which is herebyincorporated by reference). The support was then thoroughly washed indry acetone and dried at 80° C. in a vacuum desiccator.

B. Derivatization of Silanized Solid Support with Functional Groups(e.g., Carboxyl or Amino Groups)

When the silanization reagent was APTS, the desired amino functionalitywas introduced directly. Other functional groups can be introduced,either by choosing an appropriate silanization reagent primer thatalready contains the functional group (e.g., 3-(trimethoxysilyl)propylmethacrylate to functionalize the surface with a polymerizable acrylate,(M. Glad, et al., “Use of Silane Monomers Imprinting and EnzymeEntrapment in Polysiloxane-coated Porous Silica,” J. Chromatogr.347:11-23 (1985); E. Hedborg, et al., “Some Studies ofMolecularly-imprinted Polymer Membranes in Combination with Field-effectDevices,” Sensors and Actuators A 37-38:796-799 (1993); and M. Kempe, etal., “An Approach Towards Surface Imprinting Using the EnzymeRibonuclease A,” J. Mol. Recogn. 8:35-39 (1995), which are herebyincorporated by reference), or by reacting the amino-functionalizedsurface with a reagent that contains the desired functional group (e.g.,after localized light-directed photodeprotection of protected aminogroups used in photolithography, (Fodor, et al., “Light-Directed,Spatially Addressable Parallel Chemical Synthesis,” Science, 251:767-773(1991); Fodor, et al., “Multiplexed Biochemical Assays with BiologicalChips,” Nature, 364:555-556 (1993), which are hereby incorporated byreference)).

C. Activation of Functional Groups

The functional group on the solid support was an amino group. Using aprefabricated mask with a 5×5 array of dots that have a diameter of 1mm, and that are 3.25 mm apart, small amounts (typically 0.2 to 1.0 μl)of a solution containing 70 mg/ml disuccinimidyl adipate ester (Hill, etal., “Disuccinimidyl Esters as Bifunctional Crosslinking Reagents forProteins,” FEBS Lett, 102:282-286 (1979); Horton, et al., “CovalentImmobilization of Proteins by Techniques which Permit SubsequentRelease,” Methods Enzymol., pp. 130-141 (1987), which are herebyincorporated by reference) in anhydrous dimethylformamide (“DMF”);Aldrich, Milwaukee, Wis.), amended with 1-2% triethylamine (to scavengethe acid that is generated), were manually applied to the solid supportusing a Gilson P-10 pipette. After application, the reaction was allowedto proceed for 30 min at room temperature in a hood, after which anotherloading of disuccinimidyl adipate ester was applied. After a totalreaction time of 1 hour, the support was washed with anhydrous DMF anddried at room temperature in a vacuum desiccator.

In case the functional group is a carboxyl group, the solid support canbe reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (“EDC”). Frank, et al., “Simultaneous Multiple PeptideSynthesis Under Continuous Flow Conditions on Cellulose Paper Discs asSegmental Solid Support,” Tetrahedron, 44:6031-6040 (1988), which ishereby incorporated by reference. Prior to this reaction, the surface ofthe solid support was protonated by a brief treatment with 0.1 N HCl.Using the above described prefabricated mask, small amounts (0.2 to 1.0μl) of a fresh solution containing 1 M EDC (Sigma, St. Louis, Mo.), 1 mMof 5′ amino-modified oligonucleotide and 20 mM KH₂PO₄, pH=8.3, wasmanually applied to the solid support. The reaction was allowed toproceed for 1 hour, after which the support was washed with distilledwater and dried at room temperature in a vacuum desiccator.

D. Coupling of Amino-Functionalized Capture Oligonucleotides to thePreactivated Solid Support

For supports other than EDC-activated solid supports, small amounts (0.2to 1.0 μl) of 1 nmol/μl 5′ amino-modified oligonucleotides (i.e. thesequences in Table 2) in 20 mM KH₂PO₄, pH 8.3, were manually applied tothe activated support, again using the prefabricated mask describedabove. The reaction was allowed to proceed for 1 hour at roomtemperature.

E. Quenching of Remaining Reactive Groups on the Solid Support

In order to prevent the reaction products from being nonspecificallycaptured on the solid support in a capture probe-independent way, it maybe necessary to quench any remaining reactive groups on the surface ofthe solid support after capture of the complementary oligonucleotideprobes. Hereto, the support was incubated for 5 min at room temperaturein 0.1 N sodium hydroxide. Alternatively, quenching can be performed in0.2 M lysine, pH=9.0. After quenching, the support was washed with 0.1 Nsodium phosphate buffer, pH 7.2, to neutralize the surface of thesupport. After a final wash in distilled water the support was dried andstored at room temperature in a vacuum desiccator.

Example 2 Design of the Assay System

A semi-automated custom-designed assay system was made for testinghybridizations and subsequent washings of captured oligonucleotideprobe-capture oligonucleotide hybrids in a high-throughput format usingthe GeneAmp In Situ PCR System 1000™ (Perkin Elmer, Applied BiosystemsDivision, Foster City, Calif.) (G. J. Nuovo, PCR in situ Hybridization,New York: Raven Press (2nd ed. 1994), which is hereby incorporated byreference). A general flowchart of the system is shown in FIG. 27. Thesystem consists of a flow-through hybridization chamber which isconnected via a sample loading device and a multiple port system to abattery of liquid reservoirs, and to a waste reservoir. The fluiddelivery is controlled by a pump. The pump was placed at the end of theassembly line and operated under conditions to maintain a light vacuumto prevent leakage and contamination of the system. Since thehybridization chamber and the liquid reservoirs were designed to fitprecisely within the GeneAmp In Situ PCR System 1000™, temperatures canbe accurately controlled and maintained during the hybridization andwashing steps of the assay.

The individual parts of the system are described in detail in thefollowing section:

A. Hybridization Chamber

The hybridization chamber is an in situ PCR reagent containment systemthat has been modified to accommodate flow-through characteristics. Thecontainment system is comprised of a glass microscope slide(76×25×1.2±0.02 mm) and a silicone rubber diaphragm, which has beenclamped to the slide by a thin stainless steel clip. The inside oval rimof the metal clip compresses the edges of the silicon disc against theslide with enough force to create a water and gas-tight seal ensuringthe containment of hybridization probes and washing liquids. The volumeof the containment is approximately 50 μl. The array of immobilizedcapture oligonucleotides is contained in the central area of the slide(approximately 13 mm×15 mm) which is covered by the silicon disc. Theassembly of the different parts is facilitated by an assembly tool whichis provided by the manufacturer of the in situ PCR system. Onceassembled, an inlet and outlet of the hybridization chamber is createdby insertion of two 25 G¾ needles with 12″ tubing and multiple sampleluer adapter (Becton Dickinson, Rutherford, N.J.). The needles areinserted in a diagonal manner to assure an up-and-across flow patternduring washing of the probe-target hybrids.

B. Liquid Reservoirs

Reservoirs containing different washing solutions were custom-designedto fit into the vertical slots of the thermal block of the GeneAmp InSitu PCR System 1000™. Each reservoir consists of two glass microscopechamber slides (25×75×1 mm) containing prefabricated silicone gaskets(Nunc, Inc., Napierville, Ill.), which were glued to each other usingsilicone sealant (Dow Corning, Midland, Mich.). An outlet was created byinsertion of a 21 G ¾″ needle with 12″ long tubing and multiple sampleluer adapter (Becton Dickinson, Rutherford, N.J.) through the siliconegasket. A second 21 G ¾″ needle without tubing (Becton Dickinson,Franklin Lakes, N.J.) was inserted through the silicone gasket to createan air inlet. The liquid reservoirs are leak-free and fit preciselywithin the slots of the thermal block, where they are clamped againstthe metal fins to assure good heat transfer to the contained liquid. Thevolume of each reservoir is approximately 2 ml.

C. Multi Port System and Sample Loading Device

Liquid reservoirs, sample loading device and hybridization chamber areconnected through a multiple port system that enables a manuallycontrolled unidirectional flow of liquids. The system consists of aseries of 3-way nylon stopcocks with luer adapters (Kontes ScientificGlassware/Instruments, Vineland, N.J.) that are connected to each otherthrough male-female connections. The female luer adapters from theliquid reservoirs are connected to the multi port female luer adaptersvia a male-to-male luer adapter coupler (Biorad, Richmond, Calif.). Thesample loading device is placed in between the ports connected to theliquid reservoirs and the port connected to the hybridization chamber.It consists of a 1 ml syringe (Becton Dickinson, Franklin Lakes, N.J.)that is directly connected via a luer adapter to the multi port system.The flow of liquids can be controlled manually by turning the handles onthe stopcocks in the desired direction.

D. Waste Reservoir

The outlet tubing from the hybridization chamber is connected to a wastereservoir which consists of a 20 ml syringe with luer adapter (BectonDickinson, Franklin Lakes, N.J.) in which the plunger has been securedat a fixed position. A connection to the pump is established byinsertion of a 21 G ¾″ needle with 12″ long tubing and multiple sampleluer adapter through the rubber gasket of the plunger. When the pump isactivated, a slight vacuum is built up in the syringe which drives theflow of liquids from the liquid reservoirs through the hybridizationchamber to the waste reservoir.

E. Pump

A peristaltic pump P-1 (Pharmacia, Piscataway, N.J.) was used to controlthe flow of liquids through the system. It was placed at the end of theassembly line in order to maintain a slight vacuum within the system.The inlet tubing of the pump was connected to the outlet tubing of thewaste reservoir via a 3-way nylon stopcock. By this construction releaseof the vacuum within the waste reservoir is established enabling itsdraining by gravity.

Example 3 Hybridization and Washing Conditions

In order to assess the capture specificity of different captureoligonucleotides, hybridization experiments were carried out using twocapture oligonucelotide probes that had 3 out of 6 tetramers (i.e., 12out of 24 nucleotides) in common. This example represents the mostdifficult case to distinguish between different captureoligonucleotides. In general, other capture oligonucleotides would beselected that would have fewer tetramers in common to separate differentamplification products on an addressable array.

Typically, 10 pmol of each of the oligonucleotides comp 12 and comp 14(see Table 3) were 5′ end labeled in a volume of 20 μl containing 10units of T4 polynucleotide kinase (New England Biolabs, Beverly, Mass.),2.22 MBq (60 μCi) [γ-³²P] ATP, 50 mM Tris-HCl, pH 8, 10 mM MgCl₂, 1 mMEDTA, and 10 mM dithiothreitol, according to a slightly modifiedstandard procedure described in the literature. Unincorporatedradioactive nucleotides were removed by filtration over a columncontaining superfine DNA grade Sephadex G-25 (Pharmacia, Piscataway,N.J.). The Sephadex was preswollen overnight at 4° C. in 10 mM ammoniumacetate. The labeled oligonucleotide probes were dried in vacuum anddissolved in hybridization solution (0.5 M Na₂HPO₄ [pH 7.2], 1%crystalline grade BSA, 1 mM EDTA, 7% SDS). The specific activity of thelabeled oligonucleotide probes comp 12 and comp 14 was 2.86×10⁶ cpm/pmoland 2.43×10⁶ cpm/pmol, respectively.

TABLE 3 Oligonucleotides used (5′ to 3′) 12 Aminolink-spacer 18-ATC GGGTAG GTA ACC TTG CGT GCG (SEQ ID NO: 13) 14 Aminolink-spacer 18-GGT AGGTAA CCT ACC TCA GCT GCG (SEQ ID NO: 14) comp 12 CGC ACG CAA GGT TAC CTACCC GAT (SEQ ID NO: 15) comp 14 CGC AGC TGA GGT AGG TTA CCT ACC (SEQ IDNO: 16)

Four hundred picomoles of amino-linked capture oligonucleotides 12 and14 (see Table 3) were deposited and reacted both on carboxyl derivatizedand amino derivatized glass microscope slides as described in theprevious section. The capture oligonucleotides were immobilized in a 2×2matrix array, in such a way that hybridization with the complementaryoligonucleotide probe comp 12 would result in a positive signal for thetop-left and bottom-right diagonal positions, while hybridization withthe complementary oligonucleotide probe comp 14 would result in apositive signal for the bottom-left and top-right diagonal positions.

Radiolabeled oligonucleotide probes comp 12 and comp 14 (see Table 3)were dissolved in hybridization solution at a concentration of 2.5pmol/100 μl and 4.1 pmol/100 μl, respectively. The hybridizationsolutions were amended with 5 μl of a 2% bromophenol blue marker tofacilitate the visual monitoring of the probes during their transportthrough the assay system. One hundred microliters of radiolabeled probewas then injected and pumped into the hybridization chamber.Hybridizations were performed for 15 min at 70° C.

After hybridization, the hybridization chamber was sequentially washedwith 2×2 ml of low stringency wash buffer (2×SSC buffer contains 300 mMsodium chloride and 30 mM sodium citrate), 0.1% sodium dodecylsulfate(“SDS”)) and 2×2 ml of high stringency wash buffer (0.2×SSC, 0.1% SDS)at 70° C. (1×SSC buffer contains 150 mM sodium-chloride and 15 mM sodiumcitrate).

Example 4 Detection of Captured Oligonucleotide Probes

After washing the capture oligonucleotide-oligonucleotide probe hybrids,silicon discs, needles and metal cover clips were removed from the glassmicroscope slides, and remaining liquid was absorbed using Kimwipes(Kimberly-Clark, Roswell, Ga.). The captured oligonucleotide probes werevisualized and quantified using a phosphorimager (Molecular Dynamics,Sunnyvale, Calif.). After 21 hours of exposure of the glass microscopeslide to a phosphorimager screen, data were collected for the differentsolid supports that were tested. The images that were obtained are shownin FIG. 28. Quantitative data are shown in Tables 4A and 4B.

Under the conditions that were used, the signals and cross-reactivitydata that were obtained for the NH₂-functionalized slides were betterthan those obtained for the COOH-functionalized slides.

TABLE 4A Quantification of captured oligonucleotide probe 12Oligonucleotide Oligonucleotide Average Functional probe at captureprobe at capture cross group oligonucleotide 12 oligonucleotide 14 reac-on slide Probe (pic)* (amol) (pic)* (amol) tivity —COOH 12 105,333 9.00.37 —COOH 12 55,957 4.8 —COOH 12 36,534 3.1 —COOH 12 23,707 2.0 —NH₂ 12353,569 30 0.015 —NH₂ 12 10,421,092 889 —NH₂ 12 64,999 5.5 —NH₂ 1295,414 8.1 *pic = relative phosphorimager counts

TABLE 4B Quantification of captured oligonucleotide probe 14Oligonucleotide Oligonucleotide probe probe at capture at captureFunctional oligonucleotide oligonucleotide Average group 12 14 cross onslide Probe (pic)* (amol) (pic)* (amol) reactivity —COOH 14 35,610 4.00.19 —COOH 14 43,362 4.9 —COOH 14 5,587 0.6 —COOH 14 9,379 1.1 —NH₂ 14245,973 28 0.049 —NH₂ 14 115,529 13 —NH₂ 14 9,775 1.1 —NH₂ 14 8,065 0.9*pic = relative phosphorimager counts

Example 5 Optimizing Immobilization Parameters of CaptureOligonucleotides

Polymer was deposited on slides using a literature procedure. Barnard,et al., “A Fibre-optic Sensor With Discrete Sensing Sites,” Nature353:338-40 (1991); Bonk, et al., “Fabrication of Patterned Sensor ArraysWith Aryl Azides on a Polymer-coated Imaging Optical Fiber Bundle,”Anal. Chem. 66:3319-20 (1994); Smith, et al.,“Poly-N-acrylylpyrrolidone—A New Resin in Peptide Chemistry,” Int. J.Peptide Protein Res. 13:109-12 (1979), which are hereby incorporated byreference.

Four hundred picomoles of amino-linked capture oligonucleotides 12 and14 (see Table 3) were deposited and reacted in a 2×2 pattern to a glassmicroscope slide that contained 4 identical photo-deposited polymerspots. The oligonucleotides were spotted in such a way thathybridization with the complementary oligonucleotide probe comp 12 wouldresult in a positive signal for the top and bottom positions, whilehybridization with the complementary oligonucleotide probe comp 14 wouldresult in a positive signal for the left and right positions.

Radiolabeled oligonucleotide probe comp 12 (see Table 3) was dissolvedin hybridization solution at a concentration of 2.4 pmol/100 μl.

Bromophenol Blue Marker

(5 μl of a 2% solution) was added to the hybridization solution tofacilitate the monitoring of the probe during its transport through thesystem.

One hundred microliters of radiolabeled probe comp 12 was pumped intothe hybridization chamber. Hybridization was performed for 15 min at 70°C. After hybridization, the hybridization chamber was sequentiallywashed with 3×1 ml of low stringency wash buffer (2×SSC, 0.1% SDS) and3×1 ml of high stringency wash buffer (0.2×SSC, 0.1% SDS) at 70° C.

After 24 hours of exposure of the glass microscope slide to aphosphorimager screen, data were collected for all the different slidesthat were tested. The images that were obtained are shown in FIG. 29.Quantitative data are shown in Table 5.

TABLE 5 Quantification of captured oligonucleotide probes probe 12 probe12 Crosslinker Percentage crosslinker (pic)* (amol) EGDMA 2 1,055,100 801,390,499 106 HDDMA 2 633,208 48 286,9371 218 EGDMA 4 4,449,001 3382,778,414 211 EGDMA = ethylene glycol dimethacrylate HDDMA = hexane dioldimethacrylate *pic = relative phosphorimager counts

The immobilization chemistry allows for the use of tailor-made specialtypolymer matrices that provide the appropriate physical properties thatare required for efficient capture of nucleic acid amplificationproducts. The specificity of the immobilized capture oligonucleotideshas been relatively good compared to current strategies in which singlemismatches, deletions, and insertions are distinguished by differentialhybridization (K. L. Beattie, et. al. “Advances in Genosensor Research,”Clin. Chem. 41:700-06 (1995), which is hereby incorporated byreference). Finally, it has been demonstrated that the assay system ofthe present invention enables the universal identification of nucleicacid oligomers.

Example 6 Capture of Addressable Oligonucleotide Probes to Solid Support

Polymer-coated slides were tested for their capture capacity ofaddressable oligonucleotide probes following different procedures forimmobilization of capture oligonucleotides. After being silanized with3-(trimethoxysilyl) propyl methacrylate, monomers were then polymerizedon the slides. In one case, a polymer layer having COOH functionalgroups was formed with a polyethylene glycol-containing crosslinker. Inthe other case, a polyethylene glycol-methacrylate monomer waspolymerized onto the slide to form OH functional groups. The slides withthe COOH functional groups were activated using the EDC-activationprocedure of Example 1.

The slide with OH functional groups was activated overnight at roomtemperature by incubation in a tightly closed 50 ml plastic disposabletube (Corning Inc., Corning, N.Y.) containing 0.2 M1,1′-carbonyldiimidazole (“CDI”) (Sigma Chemical Co., St. Louis, Mo.) in“low water” acetone (J. T. Baker, Phillipsburg, N.J.). The slide wasthen washed with “low water” acetone, and dried in vacuum at roomtemperature.

Amino-linked capture oligonucleotide 14 was manually spotted onpremarked locations on both sides (4 dots per slide). The reactions wereperformed in a hood, and the amount of oligonucleotide that was spottedwas 2×0.2 μl (0.8 nmol/μl). The total reaction time was 1 hr. The slideswere then quenched for 15 min by the application of few drops ofpropylamine on each of the premarked dots. After quenching, the slideswere incubated for 5 min in 0.1 N sodium phosphate buffer, pH 7.2,washed in double distilled H₂O, and dried in vacuum.

The complementary capture oligonucleotides on the slides were hybridizedwith radioactively labeled oligonucleotide probe comp 14 (Table 3). Onehundred microliters radiolabeled oligonucleotide probe comp 14 (2.8pmol; 6,440,000 cpm/pmol) were pumped into the hybridization chamber.Hybridization was performed for 15 min at 70° C. in 0.5 M Na₂HPO₄ [pH7.2], 1% crystalline grade BSA, 1 mM EDTA, 7% SDS. After hybridization,the hybridization chamber was sequentially washed with 2×2 ml of lowstringency wash buffer (2×SSC, 0.1% SDS) and 2×2 ml of high stringencywash buffer (0.2×SSC, 0.1% SDS) at 70° C. (1 SSC buffer contains 150 mMsodium chloride and 15 mM sodium citrate).

After 30 min of exposure of the glass microscope slide to aphosphorimager screen, data were collected for both slides. After 30minutes of exposure of the glass microscope slides to a phosphorimagerscreen data were collected. See Table 6 and FIG. 30.

TABLE 6 Quantification of capture oligonucleotide probe 14 on OH-functionalized slides Functional Oligonucleotide probe at group captureoligonucleotide 14 on glass slide Probe (pic)* (fmol) —OH 14 1,864,87910.9 —OH 14 1,769,403 10.3 *pic = relative phosphorimager countsIn this test, better results were obtained with the slide coated withthe polymer containing OH functional groups than with the slide coatedwith the polymer containing COOH functional groups.

With previously prepared (poly HEMA)-containing polymers that werepolymerized with 20% amine-containing monomers and crosslinked with 4%EGDMA or HDDMA, it was possible to capture about 275 amol ofradioactively labelled ligated product sequence (which could only bevisualized after 23 hours of exposure to a phosphorimager screen (Table5)). Using the polyethylene-methacrylate polymer formulations, it waspossible to capture about 10.6 fmoles of ligated product sequence. Thesignal could be detected after 30 mm of exposure.

Example 7 Detection of Captured Oligonucleotides Using a MembraneSupport

In order to assess the capture specificity of different captureoligonucleotides using a membrane support, hybridization experimentswere carried out using the capture oligonucleotide probes 12 and 14(Table 3).

Strips of OH-functionalized nylon membrane (Millipore, Bedford, Mass.)were soaked overnight in a 0.2 M solution of carbonyldiimidazole in “lowwater” acetone. The strips were washed in acetone and dried in vacuo.Two volumes of 0.2 μl (1 mM) capture oligonucleotides 12 and 14 in 20 mMK₂HPO₄, pH 8.3, (Table 3) were loaded on the membrane using a specialblotting device (Immunetics, Cambridge, Mass.). Complementaryoligonucleotide probes were radioactively labeled as described inExample 3. The oligonucleotide probes were dried in vacuo and taken upin 200 μl hybridization buffer (0.5 M Na₂HPO₄ [pH 7.2], 1% crystallinegrade BSA, 1 mM EDTA, 7% SDS). Membranes were prehybridized in 800 μlhybridization buffer for 15 min at 60° C. in 1.5 ml Eppendorf tubes in aHybaid hybridization oven. The tubes were filled with 500 μl of inertcarnauba wax (Strahl & Pitsch, Inc., New York, N.Y.) to reduce the totalvolume of the hybridization compartment. After prehybridization, 200 μlof radiolabeled probe was added. The membranes were hybridized for 15min at 60° C. After hybridization, the membranes were washed at 60° C.,twice for 15 min with 1 ml of low stringency wash buffer (2×SSC, 0.1%SDS), and twice for 15 min with 1 ml of high stringency wash buffer(0.2×SSC, 0.1% SDS). The captured oligonucleotide probes were quantifiedusing a phosphorimager (Molecular Dynamics, Sunnyvale, Calif.). After 45min of exposure to a phosphorimager screen, data were collected. Theresults are shown in Table 7, where the activities of captureoligonucleotides 12 and 14 are 112 pic/amol and 210 pic/amol,respectively.

TABLE 7 Quantification of captured oligonucleotides on membranes Func-tional group Oligonucleotide Oligonucleotide Average on probe at captureprobe at capture cross mem- oligonucleotide 12 oligonucleotide 14 reac-brane Probe (pic)* (fmol) (pic)* (fmol) tivity —OH 12 13,388,487 119.5337,235 3.01 0.025 —OH 12 13,299,298 118.7 —OH 14 179,345 0.85 1,989,8769.48 0.071 —OH 14 3,063,387 14.59 *pic = relative phosphorimager counts

Hybridization temperatures and hybridization times were further exploredin a series of similar experiments. The data shown in Table 8 (where theactivities of capture oligonucleotides 12 and 14 are 251 pic/amol and268 pic/amol, respectively) represent the results obtained with thefollowing conditions: 15 min prehybridization at 65° C. in 800 μlhybridization buffer; 15 min hybridization at 65° C. in 1 mlhybridization buffer; 2× washings for 5 min at 65° C. with 1 ml of lowstringency wash buffer; and 2× washings for 5 min at 65° C. with 1 ml ofhigh stringency wash buffer.

TABLE 8 Quantification of captured oligonucleotides on membranes Func-tional group Oligonucleotide Oligonucleotide Average on probe at captureprobe at capture cross mem- oligonucleotide 12 oligonucleotide 14 reac-brane Probe (pic)* (fmol) (pic)* (fmol) tivity —OH 12 41,023,467 163.4541,483 2.16 0.015 —OH 12 31,868,432 127.0 —OH 14 294,426 1.1019,673,325 73.41 0.016 —OH 14 18,302,187 68.29 *pic = relativephosphorimager counts

The data shown in Table 9 (where the activities of captureoligonucleotides 12 and 14 are 487 pic/amol and 506 pic/amol,respectively) represent the results obtained with the followingconditions: 15 min prehybridization at 70° C. in 150 μl hybridizationbuffer; 15 min hybridization at 70° C. in 200 μl hybridization buffer;2× washings for 5 min at 70° C. in 800 μl of low stringency wash buffer;and 2× washings for 5 min at 70° C. in 800 μl of high stringency washbuffer.

TABLE 9 Quantification of captured oligonucleotides on membranesOligonucleotide Oligonucleotide Functional probe at capture probe atcapture Average group on oligonucleotide 12 oligonucleotide 14 crossmembrane Probe (pic)* (fmol) (pic)* (fmol) reactivity —OH 12 34,648,38571.15 1,158,832 2.38 0.027 —OH 12 52,243,549 107.28 —OH 14 1,441,6912.85 56,762,990 112.18 0.028 —OH 14 45,769,158 90.45 *pic = relativephosphorimager counts

The data shown in Table 10 represent the results obtained with thefollowing conditions: 15 min prehybridization at 70° C. in 150 μlhybridization buffer; 5 min hybridization at 70° C. in 200 μlhybridization buffer; 2× washings for 5 min at 70° C. with 800 μl of lowstringency wash buffer; and 2× washings for min at 70° C. with 800 μl ofhigh stringency wash buffer.

TABLE 10 Quantification of captured oligonucleotides on membranes.Oligonucleotide probe Oligonucleotide Functional at capture probe atcapture Average group on oligonucleotide 12 oligonucleotide 14 crossmembrane Probe (pic)* (fmol) (pic)* (fmol) reactivity —OH 12 26,286,18853.98 389,480 0.80 0.013 —OH 12 34,879,649 71.62 —OH 14 539,486 1.0745,197,674 89.32 0.011 —OH 14 54,409,947 107.53 *pic = relativephosphorimager counts

The data shown in Table 11 represent the results obtained with thefollowing conditions: 5 min prehybridization at 70° C. in 150 μlhybridization buffer; 1 min hybridization at 70° C. in 200 μlhybridization buffer; 2× washings for 2 min at 70° C. with 800 μl of lowstringency wash buffer; and 2× washings for 5 min at 70° C. with 800 μlof high stringency wash buffer.

TABLE 11 Quantification of captured oligonucleotides on membranesOligonucleotide Oligonucleotide Functional probe at capture probe atcapture Average group on oligonucleotide 12 oligonucleotide 14 crossmembrane Probe (pic)* (fmol) (pic)* (fmol) reactivity —OH 12 5,032,83510.33 56,777 0.12 0.012 —OH 12 4,569,483 9.38 —OH 14 540,166 1.0741,988,355 82.98 0.017 —OH 14 20,357,554 40.23 *pic = relativephosphorimager counts

These data demonstrate that hybridization of the capture oligonucleotideprobes to their complementary sequences was specific. In comparison withthe previous experiments performed with glass slides, significantlygreater amounts (i.e., fmol quantities compared to amol quantities) ofoligonucleotide probes were reproducibly captured on the membranesupports. For these two very closely-related capture oligonucleotideprobes, average cross-reactivity values of about 1% could be obtained.However, for other pairs of capture oligonucleotides in the array, thesevalues would be significantly better. In general, such values cannot beachieved by using existing methods that are known in the art, i.e., byallele-specific oligonucleotide hybridization (“ASO”) or by differentialhybridization methods, such as sequencing by hybridization (“SBH”).

Example 8 Cleaning Glass Surfaces

Glass slides (Fisher Scientific, Extra thick microslides, frosted cat.#12-550-11) were incubated in conc. aq. NH₄OH—H₂O₂—H₂O (1:1:5, v/v/v) at80° C. for 5 min and rinsed in distilled water. A second incubation wasperformed in conc. aq HCl—H₂O₂—H₂O (1:1:5, v/v/v) at 80° C. for 5 min.See U. Jönsson, et al., “Absorption Behavior of Fibronectin on WellCharacterized Silica Surfaces,” J. Colloid Interface Sci. 90:148-163(1982), which is hereby incorporated by reference. The slides wererinsed thoroughly in distilled water, methanol, and acetone, and wereair-dried at room temperature.

Example 9 Silanization with 3-methacryloyloxypropyltrimethoxysilane

Cleaned slides, prepared according to Example 8, were incubated for24-48 h at room temperature in a solution consisting of 2.6 ml of3-methacryloyloxypropyltrimethoxysilane (Aldrich Chemical Company, Inc.Milwaukee, Wis. cat.# 23,579-2), 0.26 ml of triethylamine, and 130 ml oftoluene. See E. Hedborg, et. al., Sensors Actuators A, 37-38:796-799(1993), which is hereby incorporated by reference. The slides wererinsed thoroughly in acetone, methanol, distilled water, methanol again,and acetone again, and were air-dried at room temperature. See FIG. 31.

Example 10 Silanization with Dichlorodimethylsilane

Cleaned slides, prepared according to Example 8, were incubated for 15min at room temperature in a solution containing 12 ml ofdichlorodimethylsilane and 120 ml of toluene. The slides were rinsedthoroughly in acetone, methanol, distilled water, methanol again, andacetone again and were air-dried.

Example 11 Polymerization of Poly(ethylene glycol)methacrylate withMethacrylate-Derivatized Glass

2.2 g of poly(ethylene glycol)methacrylate (Aldrich Chemical Company,Inc. Milwaukee, Wis. cat.# 40,953-7) (average M˜306 g/mol) and 50 mg of2,2′-azobis(2-methylpropionitrile) in 3.5 ml of acetonitrile were cooledon ice and purged with a stream of argon for 3 min. The next steps wereperformed in a glovebox under argon atmosphere. 5-15 drops of thepolymerization mixture were placed on a methacrylate-derivatized glassslide, prepared according to Examples 8 and 9. Themethacrylate-derivatized glass slide and the polymerization mixture werecovered by a second glass slide which had been silanized according toExample 10, and the two glass slides were pressed together and fixedwith clips. The slides were subsequently transferred to a vacuumdesiccator. The polymerization was thermolytically initiated at 55° C.,or photolytically at 366 nm. See FIG. 32.

Example 12 Polymerization of Acrylic Acid and TrimethylolpropaneEthoxylate (14/3 EO/OH) Triacrylate with Methacrylate-Derivatized Glass

0.5 g of acrylic acid (Aldrich Chemical Company, Inc. Milwaukee, Wis.cat.# 14,723-0), 1.83 g of trimethylolpropane ethoxylate (14/3 EO/OH)triacrylate (Aldrich Chemical Company, Inc. Milwaukee, Wis. cat.#23,579-2) and 50 mg of 2,2′-azobis(2-methylpropionitrile) in 3.5 ml ofacetonitrile were cooled on ice and purged with a stream of argon for 3min. The next steps were performed in a glovebox as described in Example11. The slides were subsequently transferred to a vacuum desiccator andpolymerized as described in Example 11. See FIG. 33.

Example 13 Polymerization of Poly(ethylene glycol)methacrylate andTrimethylolpropane Ethoxylate (14/3 EO/OH) Triacrylate withMethacrylate-Derivatized Glass

0.55 g of poly(ethylene glycol)methacrylate (Aldrich Chemical Company,Inc. Milwaukee, Wis. cat.# 40,953,7), 1.64 g of trimethylolpropaneethoxylate (14/3 EO/OH triacrylate (Aldrich Chemical Company, Inc.Milwaukee, Wis. cat.# 23,579-2), and 50 mg of2,2′-azobis(2-methylpropionitrile) in 3.5 ml of acetonitrile were cooledon ice and purged with a stream of argon for 3 min. The next steps wereperformed in a glove-box as described in Example 11. The slides weresubsequently transferred to a vacuum desiccator and polymerized asdescribed in Example 11. See FIG. 34.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such details are solely for thatpurpose and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A method for identifying a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations, saidmethod comprising: providing a sample potentially containing targetnucleotide sequences differing by one or more single-base changes,insertions, deletions, or translocations; providing a plurality ofoligonucleotide probe sets, each set characterized by (a) a firstoligonucleotide probe, having a target-specific portion and anaddressable array-specific portion and (b) a second oligonucleotideprobe, having a target-specific portion and a detectable reporter label;providing a ligase; blending the sample, the plurality ofoligonucleotide probe sets, and the ligase to form a mixture; subjectingthe mixture to one or more ligase detection reaction cycles to form aligated product sequence containing (a) the addressable array-specificportion, (b) the target-specific portions, and (c) the detectablereporter label, if their respective target nucleotide is present in thesample; providing a solid support with capture oligonucleotidesimmobilized at particular sites, wherein the capture oligonucleotides,each having greater than 16 nucleotides, have nucleotide sequencescomplementary to the addressable array-specific portions with thecapture oligonucleotides hybridizing to their complementary addressablearray specific portion under uniform hybridization conditions, whereinadjacent capture oligonucleotides on the solid support which arecomplementary to the addressable array-specific portions correspondingto single-base changes, insertions, deletions, or translocations atdifferent locations in the target nucleotide sequences differ in theirnucleotide sequences, when aligned to each other, by at least 25% of thenucleotides to minimize cross hybridization between non-complementarycapture oligonucleotides and addressable array-specific portions, and,wherein the solid support and the capture oligonucleotides form anaddressable array; contacting the mixture, after said subjecting, withthe solid support under conditions effective to hybridize theaddressable array-specific portions to the capture oligonucleotides in abase-specific manner, thereby capturing the addressable array-specificportions on the solid support at the site with the complementary captureoligonucleotide; and detecting and distinguishing the reporter labels ofligated product sequences captured to the solid support at particularsites, thereby indicating the presence of target nucleotide sequences inthe sample, wherein the oligonucleotide probe sets are configured sothat the addressable array-specific portion is comprised of a nucleotidesequence which is distinct from that of the target-specific portions, inorder to prevent hybridization between the target-specific portions andthe capture oligonucleotides as well as between the target nucleotidesequence and the addressable array-specific portion.
 2. The methodaccording to claim 1, wherein the oligonucleotide probe sets aresuitable for ligation together at a ligation junction when hybridizedadjacent to one another on a corresponding target nucleotide sequencedue to perfect complementarity at the ligation junction, but, when theoligonucleotide probe sets are hybridized to any other nucleotidesequence present in the sample, have a mismatch at a base at theligation junction which interferes with such ligation.
 3. The methodaccording to claim 2, wherein the mismatch is at the 3′ base at theligation junction.
 4. The method according to claim 1, wherein theoligonucleotide probes in a set are suitable for ligation together at aligation junction when hybridized adjacent to one another on acorresponding target nucleotide sequence due to perfect complementarityat the ligation junction, but, when the oligonucleotide probes in theset are hybridized to any other nucleotide sequence present in thesample, there is a mismatch at a base adjacent to a base at the ligationjunction which interferes with such ligation.
 5. The method according toclaim 4, wherein the mismatch is at the base adjacent to the 3′ base atthe ligation junction.
 6. The method according to claim 1, wherein thesample potentially contains unknown amounts of one or more of aplurality of target sequences with a plurality of sequence differences,said method further comprising: quantifying, after said detecting, theamount of the target nucleotide sequences in the sample by comparing theamount of captured ligated product sequences generated from the samplewith a calibration curve of captured ligated product sequences generatedfrom samples with known amounts of the target nucleotide sequence. 7.The method according to claim 1, wherein the sample potentially containsunknown amounts of one or more of a plurality of target nucleotidesequences with a plurality of sequence differences, said method furthercomprising: providing a known amount of one or more marker targetnucleotide sequences; providing a plurality of marker-specificoligonucleotide probe sets, each set characterized by (a) a firstoligonucleotide probe, having an oligonucleotide target-specific portioncomplementary to the marker target nucleotide sequence and anaddressable array-specific portion complementary to captureoligonucleotides on the solid support, and (b) a second oligonucleotideprobe, having an oligonucleotide target-specific portion complementaryto the marker target nucleotide sequence and a detectable reporterlabel, wherein said blending comprises blending the sample, the markertarget nucleotide sequences, the plurality of oligonucleotide probesets, the plurality of marker-specific oligonucleotide probe sets, andthe ligase to form a mixture; detecting the reporter labels of theligated marker-specific oligonucleotide sets captured on the solidsupport at particular sites, thereby indicating the presence of one ormore marker target nucleotide sequences in the sample; and quantifyingthe amount of target nucleotide sequences in the sample by comparing theamount of captured ligated product generated from the known amount ofmarker target nucleotide sequences with the amount of captured otherligated product.
 8. The method according to claim 7, wherein the one ormore marker target nucleotide sequences differ from the targetnucleotide sequences in the sample at one or more single nucleotidepositions.
 9. The method according to claim 8, wherein theoligonucleotide probe sets and the marker-specific oligonucleotide probesets form a plurality of oligonucleotide probe groups, each groupcomprised of one or more oligonucleotide probe sets designed fordistinguishing multiple allele differences at a single nucleotideposition, wherein, in the oligonucleotide probe sets of each group, thefirst oligonucleotide probes have a common target-specific portion, andthe second oligonucleotide probes have a differing target-specificportion which hybridize to a given allele or a marker nucleotidesequence in a base-specific manner.
 10. The method according to claim 8,wherein the oligonucleotide probe sets and the marker-specificoligonucleotide probe sets form a plurality of oligonucleotide probegroups, each group comprised of one or more oligonucleotide probe setsdesigned for distinguishing multiple allele differences at a singlenucleotide position, wherein, in the oligonucleotide probe sets of eachgroup, the second oligonucleotide probes have a common target-specificportion and the first oligonucleotide probe have differingtarget-specific portions, which hybridize to a given allele or a markernucleotide sequence in a base-specific manner.
 11. The method accordingto claim 1, wherein the sample potentially contains unknown amounts oftwo or more of a plurality of target nucleotide sequences with aplurality of sequence differences, said method further comprising:quantifying, after said detecting, the relative amount of each of theplurality of target nucleotide sequences in the sample by comparing therelative amount of captured ligated product sequences generated by eachof the plurality of target sequences within the sample, therebyproviding a quantitative measure of the relative level of two or moretarget nucleotide sequences in the sample.
 12. The method according toclaim 1, wherein multiple allele differences at two or more adjacentnucleotide positions, or at nucleotide positions which requireoverlapping oligonucleotide probe sets, in a single target nucleotidesequence or multiple allele differences at two or more adjacentnucleotide positions, or at nucleotide positions which requireoverlapping oligonucleotide probe sets, in multiple target nucleotidesequences are distinguished with oligonucleotide probe sets havingoligonucleotide probes with target-specific portions which overlap. 13.The method according to claim 1, wherein the target-specific portions ofthe oligonucleotide probe sets are configured to be successfully ligatedin the presence of their target sequences under a single set of ligasedetection reaction conditions.
 14. The method according to claim 1,wherein multiple allele differences at one or more nucleotide positionin a single target nucleotide sequence or multiple allele differences atone or more positions in multiple target nucleotide sequences aredistinguished, the oligonucleotide probe sets forming a plurality ofoligonucleotide probe groups, each group comprised of one or moreoligonucleotide probe sets designed for distinguishing multiple alleledifferences at a single nucleotide position, wherein, in theoligonucleotide probes of each group, the second oligonucleotide probeshave a common target-specific portion and the first oligonucleotideprobes have differing target-specific portions which hybridize to agiven allele in a base-specific manner, wherein, in said detecting, thelabels of ligated product sequences of each group, captured on the solidsupport at different sites, are detected, thereby indicating a presence,in the sample of one or more allele at one or more nucleotide positionin one or more target nucleotide sequences.
 15. The method according toclaim 14, wherein the oligonucleotide probes in a given set are suitablefor ligation together at a ligation junction when hybridized adjacent toone another on a corresponding target nucleotide sequence due to perfectcomplementarity at the ligation junction, but, when hybridized to anyother nucleotide sequence present in the sample, the firstoligonucleotide probe has a mismatch at a base at the ligation junctionwhich interferes with such ligation.
 16. The method according to claim14, wherein multiple allele differences at two or more adjacentnucleotide positions, or at nucleotide positions which requireoverlapping oligonucleotide probe sets, in a single target nucleotidesequence or multiple allele differences at two or more adjacentnucleotide positions, or at nucleotide positions which requireoverlapping oligonucleotide probe sets, in multiple target nucleotidesequences are distinguished with oligonucleotide probe groups havingoligonucleotide probes with target-specific portions which overlap. 17.The method according to claim 16, wherein the oligonucleotide probes ina set are suitable for ligation together at a ligation junction whenhybridized adjacent to one another on a corresponding target nucleotidesequence due to perfect complementarity at the ligation junction, but,when the oligonucleotide probes in the set are hybridized to any othernucleotide sequence present in the sample, there is a mismatch at a baseat the ligation junction which interferes with such ligation.
 18. Themethod according to claim 1, wherein multiple allele differencesconsisting of insertions, deletions, microsatellite repeats,translocations, or other DNA rearrangements at one or more nucleotidepositions which require overlapping oligonucleotide probe sets in asingle target nucleotide sequence or multiple allele differencesconsisting of insertions, deletions, microsatellite repeats,translocations, or other DNA rearrangements at one or more nucleotidepositions which require overlapping oligonucleotide probe sets inmultiple target nucleotide sequences are distinguished, theoligonucleotide probe sets forming a plurality of oligonucleotide probegroups, each group comprised of one or more oligonucleotide probe setsdesigned for distinguishing multiple allele differences selected fromthe group consisting of insertions, deletions, microsatellite repeats,translocations, and other DNA rearrangements at one or more nucleotidepositions which require overlapping oligonucleotide probe sets, wherein,in the oligonucleotide probe sets of each group, the secondoligonucleotide probes have a common oligonucleotide target-specificportion and the first oligonucleotide probes have differingoligonucleotide target-specific portions which hybridize to a givenallele in a base-specific manner, wherein, in said detecting, the labelsof ligated product sequences of each group, captured on the solidsupport at different sites, are detected, thereby indicating a presence,in the sample, of one or more allele differences selected from the groupconsisting of insertions, deletions, microsatellite repeats,translocations, and other DNA rearrangements in one or more targetnucleotide sequences.
 19. The method according to claim 18, wherein theoligonucleotide probe sets are designed for distinguishing multipleallele differences selected from the group consisting of insertions,deletions, and microsatellite repeats, at one or more nucleotidepositions which require overlapping oligonucleotide probe sets, wherein,in the oligonucleotide probe sets of each group, the secondoligonucleotide probes have a common target-specific portion, and thefirst oligonucleotide probes have differing target-specific portionswhich contain repetitive sequences of different lengths to hybridize toa given allele in a base-specific manner.
 20. The method according toclaim 1, wherein a low abundance of multiple allele differences atmultiple adjacent nucleotide positions, or at nucleotide positions whichrequire overlapping oligonucleotide probe sets, in a single targetnucleotide sequence, in the presence of an excess of normal sequence, ora low abundance of multiple allele differences at multiple nucleotidepositions which require overlapping oligonucleotide probe sets, inmultiple target nucleotide sequences, in the presence of an excess ofnormal sequence, are distinguished, the oligonucleotide probe setsforming a plurality of oligonucleotide probe groups, each groupcomprised of one or more oligonucleotide probe sets designed fordistinguishing multiple allele differences at a single nucleotideposition, wherein one or more sets within a group share common secondoligonucleotide probes and the first oligonucleotide probes havediffering target-specific portions which hybridize to a given alleleexcluding the normal allele in a base-specific manner, wherein, in saiddetecting, the labels of ligated product sequences of each groupcaptured on the solid support at different sites, are detected, therebyindicating a presence, in the sample, of one or more low abundancealleles at one or more nucleotide positions in one or more targetnucleotide sequences.
 21. The method according to claim 20, wherein theoligonucleotide probe sets are suitable for ligation together at aligation junction when hybridized adjacent to one another on acorresponding target nucleotide sequence due to perfect complementarityat the ligation junction, but, when the oligonucleotide probe sets arehybridized to any other nucleotide sequence present in the sample, thefirst oligonucleotide probes have a mismatch at a base at the ligationjunction which interferes with such ligation.
 22. The method accordingto claim 20, wherein a low abundance of multiple allele differences atmultiple adjacent nucleotide positions, or at nucleotide positions whichrequire overlapping oligonucleotide probe sets, in a single targetnucleotide sequence, in the presence of an excess of normal sequence, ora low abundance of multiple allele differences at multiple nucleotidepositions which require overlapping oligonucleotide probe sets inmultiple target nucleotide sequences, in the presence of an excess ofnormal sequence, are quantified in a sample, said method furthercomprising: providing a known amount of one or more marker targetnucleotide sequences; providing a plurality of marker-specificoligonucleotide probe sets, each set characterized by (a) a firstoligonucleotide probe having an oligonucleotide target-specific portioncomplementary to the marker target nucleotide sequence and anaddressable array-specific portion, and (b) a second oligonucleotideprobe, having an oligonucleotide target-specific portion complementaryto the marker target nucleotide sequence and a detectable reporterlabel; providing a plurality of oligonucleotide probe groups, each groupcomprised of one or more oligonucleotide probe sets or marker-specificoligonucleotide probe sets designed for distinguishing multiple alleledifferences at a single nucleotide position, including marker nucleotidesequences, wherein one or more sets within a group share a common secondoligonucleotide probe and the first oligonucleotide probes havedifferent target-specific probe portions which hybridize to a givenallele or a marker nucleotide sequence excluding the normal allele, in abase-specific manner, wherein said blending comprises blending thesample, the marker target nucleotide sequences, the plurality ofoligonucleotide probe sets, the plurality of marker-specificoligonucleotide probe sets, and the ligase to form a mixture; detectingthe reporter labels of the ligated marker-specific oligonucleotide setscaptured on the solid support at particular sites, thereby indicatingthe presence of one or more marker target nucleotide sequences in thesample; and quantifying the amount of target nucleotide sequences in thesample by comparing the amount of captured ligated products generatedfrom the known amount of marker target nucleotide sequences with theamount of other captured ligated product generated from the lowabundance unknown sample.
 23. The method according to claim 22, whereinthe oligonucleotide probe sets are suitable for ligation together at aligation junction when hybridized adjacent to one another on acorresponding target nucleotide sequence under selected conditions dueto perfect complementarity at the ligation junction, but, when theoligonucleotide probe sets are hybridized to any other nucleotidesequence present in the sample, the first oligonucleotide probes have amismatch at a base at the ligation junction which interferes with suchligation.
 24. The method according to claim 1, wherein multiple alleledifferences at one or more nucleotide position in a single targetnucleotide sequence or multiple allele differences at one or morepositions in multiple target nucleotide sequences are distinguished, theoligonucleotide sets forming a plurality of oligonucleotide probegroups, each group comprised of one or more oligonucleotide probe setsdesigned for distinguishing multiple allele differences at a singlenucleotide position, wherein, in the oligonucleotide probes of eachgroup, the first oligonucleotide probes have a common target-specificportion and the second oligonucleotide probes have differingtarget-specific portions which hybridize to a given allele in abase-specific manner, wherein, in said detecting, different reporterlabels of ligated product sequences of each group captured on the solidsupport at particular sites are detected, thereby indicating a presence,in the sample, of one or more allele at one or more nucleotide positionsin one or more target nucleotide sequences.
 25. The method according toclaim 24, wherein the oligonucleotide probe sets are suitable forligation together at a ligation junction when hybridized adjacent to oneanother on a corresponding target nucleotide sequence due to perfectcomplementarity at the ligation junction, but, when the oligonucleotideprobe sets are hybridized to any other nucleotide sequence present inthe sample, the second oligonucleotide probes have a mismatch at a baseat the ligation junction which interferes with such ligation.
 26. Themethod according to claim 24, wherein multiple allele differences at twoor more adjacent nucleotide positions, or at nucleotide positions whichrequire overlapping oligonucleotide probe sets, in a single targetnucleotide sequence, or multiple allele differences at two or moreadjacent nucleotide positions, or at nucleotide positions which requireoverlapping oligonucleotide probe sets, in multiple target nucleotidesequences are distinguished, the oligonucleotide probe groups containingoligonucleotide probes with target-specific portions which overlap. 27.The method according to claim 26, wherein the oligonucleotide probe setsare suitable for ligation together at a ligation junction whenhybridized adjacent to one another on a corresponding target nucleotidesequence due to perfect complementarity at the ligation junction, but,when the oligonucleotide probe sets are hybridized to any othernucleotide sequence present in the sample, the second oligonucleotideprobe has a mismatch at a base at the ligation junction which interfereswith such ligation.
 28. The method according to claim 1, whereinmultiple allele differences at one or more nucleotide position in asingle target nucleotide sequence or multiple allele differences at oneor more positions in multiple target nucleotide sequences aredistinguished, the oligonucleotide sets forming a plurality of probegroups, each group comprised of one or more oligonucleotide probe setsdesigned for distinguishing multiple allele differences at a singlenucleotide position, wherein, in the oligonucleotide probes of differentgroups, the second oligonucleotide probes have a common target-specificportion or the first oligonucleotide probes have a commontarget-specific portion, wherein, in said detecting, the one of aplurality of labeled ligated product sequences of each group captured onthe solid support at particular sites are detected, thereby indicating apresence of one or more allele at one or more nucleotide positions inone or more target nucleotide sequences in the sample.
 29. The methodaccording to claim 28, wherein the oligonucleotide probe sets aresuitable for ligation together at a ligation junction when hybridizedadjacent to one another on a corresponding target nucleotide sequencedue to perfect complementarity at the ligation junction but, when theoligonucleotide probe sets are hybridized to any other nucleotidesequence present in the sample, the first or second oligonucleotideprobes have a mismatch at a base at the ligation junction whichinterferes with such ligation.
 30. The method according to claim 29,wherein all possible single-base mutations for a single codon in asingle target nucleotide sequence, all possible single-base mutationsfor multiple codons in a single target nucleotide sequence, and allpossible single-base mutations for multiple codons in multiple targetnucleotide sequences are distinguished, the oligonucleotide sets forminga plurality of oligonucleotide probe groups, each group comprised of oneor more oligonucleotide probe sets designed for distinguishing allpossible single-base mutations for a single codon, wherein, in theoligonucleotide probes of each group, the second, oligonucleotide probesdiffer only in their 5′ bases at their ligation junction and containdifferent reporter labels, the first oligonucleotide probes differ onlyin their 3′ bases at their ligation junction and contain differentaddressable array-specific portions, or the first oligonucleotide probesdiffer only in their 3′ bases adjacent to the base at the ligationjunction and contain different addressable array-specific portions. 31.The method according to claim 29, wherein the oligonucleotide probe setsare suitable for ligation together at a ligation junction whenhybridized adjacent to one another on a corresponding target nucleotidesequence due to perfect complementarity at the ligation junction, but,when the oligonucleotide probe sets are hybridized to any othernucleotide sequence present in the sample, the first oligonucleotideprobes have a mismatch at the 3′ base at the ligation junction or the 3′base adjacent the base at the ligation junction or the secondoligonucleotide probes have a mismatch at the 5′ base at the ligationjunction which interferes with such ligation.
 32. The method accordingto claim 31, wherein all possible single-base mutations for a singlecodon in a single target nucleotide sequence, or all possiblesingle-base mutations for two or more adjacent codons, or at nucleotidepositions which require overlapping oligonucleotide probe sets, inmultiple target nucleotide sequences are distinguished, theoligonucleotide probe groups containing oligonucleotide probes withtarget-specific portions which overlap.
 33. The method according toclaim 28, wherein multiple allele differences at two or more adjacentnucleotide positions, or at nucleotide positions which requireoverlapping oligonucleotide probe sets, in a target nucleotide sequenceor multiple allele differences at two or more adjacent nucleotidepositions, or at nucleotide positions which require overlappingoligonucleotide probe sets, in multiple target nucleotide sequence aredistinguished, the oligonucleotide probe groups containing probes withtarget-specific portions which overlap.
 34. The method according toclaim 33, wherein oligonucleotide probes in a set are suitable forligation together at a ligation junction when hybridized adjacent to oneanother on a corresponding target nucleotide sequence due to perfectcomplementarity at the ligation junction, but, when the oligonucleotidesin the set are hybridized to any other nucleotide sequence present inthe sample, the first or second oligonucleotide probes have a mismatchat a base at the ligation junction which interferes with such ligation.35. The method according to claim 1, wherein the solid support is madefrom a material selected from the group consisting of plastic, ceramic,metal, resin, gel, glass, silicon, and composites thereof.
 36. Themethod according to claim 1, wherein the solid support is in a formselected from the group consisting of slides, discs, membranes, films,and composites thereof.
 37. The method according to claim 36, whereinthe solid support has wells, raised regions, or etched trenches.
 38. Themethod according to claim 36, wherein the solid support is in the formof a microtiter plate.
 39. The method according to claim 1, wherein thesolid support has an array of positions with the captureoligonucleotides attached to positions in the array.
 40. The methodaccording to claim 1, wherein said detecting comprises: scanning thesolid support at the particular sites and identifying if ligation of theoligonucleotide probe sets occurred and correlating identified ligationto a presence or absence of the target nucleotide sequences.
 41. Themethod according to claim 40, wherein said scanning is carried out byscanning electron microscopy, electron microscopy, confocal microscopy,charge-coupled device, scanning tunneling electron microscopy, infraredmicroscopy, atomic force microscopy, electrical conductance, andfluorescent or phosphor imaging.
 42. The method according to claim 1,wherein some of the plurality of capture oligonucleotides have identicalnucleotide sequences and different labels are used for some differenttarget nucleotide sequence.
 43. The method according to claim 1, whereinthe plurality of capture oligonucleotides each have different nucleotidesequences.
 44. The method according to claim 43, wherein each captureoligonucleotide has adjacent capture oligonucleotides separated fromadjacent capture oligonucleotides by barrier oligonucleotides to whichligated oligonucleotide probe sets will not hybridize during saidcontacting.
 45. The method according to claim 1, wherein theoligonucleotide probe sets hybridize to the target nucleotide sequencesat temperatures which are less than that at which the captureoligonucleotides hybridize to the addressable array-specific portion ofoligonucleotide probe sets.
 46. The method according to claim 1 furthercomprising: removing oligonucleotides bound to the captureoligonucleotides to permit reuse of the solid support with immobilizedcapture oligonucleotides.
 47. The method according to claim 1, whereinthe solid support includes different capture oligonucleotidesimmobilized at different sites with different capture oligonucleotidesbeing complementary to different addressable array-specific portions,whereby different oligonucleotide probe sets are captured and detectedat different sites on the solid support.
 48. The method according toclaim 1, wherein the solid support includes identical captureoligonucleotides immobilized on the solid support with the captureoligonucleotides being complementary to all the addressablearray-specific portions and the labels attached to the oligonucleotideprobe sets being different, whereby the different oligonucleotide probesets are detected and distinguished by the different labels.
 49. Themethod according to claim 1, wherein the capture oligonucleotides are inthe form of DNA or PNA.
 50. The method according to claim 1, whereinsequences differing by one or more single-base changes, insertions,deletions, or translocations are discriminated from one another duringthe ligase detection reaction and the discriminated sequences aredetected as a result of capture on the solid support.
 51. A method foridentifying a plurality of sequences differing by one or moresingle-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences comprising: producing ligationproducts from a reaction mixture, wherein said reaction mixturecomprises: a ligase; target nucleotide sequences; and oligonucleotideprobe sets, each probe set including (a) a first oligonucleotide probecomprising a first target-specific portion capable of hybridizing to acorresponding target nucleotide sequence and (b) a secondoligonucleotide probe comprising a second target-specific portioncapable of hybridizing to said corresponding target nucleotide sequence,wherein a ligation product comprising the first and the secondtarget-specific portions is produced when the first and the secondtarget-specific portions are hybridized to said corresponding targetnucleotide sequence, but is not produced when the first and the secondtarget-specific portions are hybridized with one or more mismatches to anucleotide sequence present in said reaction mixture, wherein each ofsaid ligation products comprises a ligated sequence which includes (1)the first target-specific portion of the first oligonucleotide probe ina corresponding probe set, (2) the second target-specific portion of thesecond oligonucleotide probe in said corresponding probe set, and (3) anaddressable array-specific portion, or complement thereof, that iscomprised of a nucleotide sequence that is distinct from thetarget-specific portions, in order to prevent hybridization between thetarget-specific portions and the capture oligonucleotides as well asbetween the target nucleotide sequence and the addressablearray-specific portion; capturing polynucleotides comprising saidligated sequences to a solid support, wherein each polynucleotidefurther comprises a reporter label, and the reporter label together withthe addressable array-specific portion in that polynucleotidedistinguish the ligated sequence in that polynucleotide from those inother polynucleotides with the capture oligonucleotides, each havinggreater than 16 nucleotides, hybridizing to their complementaryaddressable array specific portion under uniform hybridizationconditions, wherein adjacent capture oligonucleotides on the solidsupport which are complementary to the addressable array-specificportions corresponding to single-base changes, insertions, deletions, ortranslocations at different locations in the target nucleotide sequencesdiffer in their nucleotide sequences, when aligned to each other, by atleast 25% of the nucleotides to minimize cross hybridization betweennon-complementary capture oligonucleotides and addressablearray-specific portions; and detecting the reporter labels and theidentities of the addressable sequences in said captured polynucleotidesto indicate the presence of one or more target nucleotide sequences insaid reaction mixture.